U.S. patent number 9,791,447 [Application Number 14/247,153] was granted by the patent office on 2017-10-17 for methods of identifying senp1 inhibitors.
This patent grant is currently assigned to City of Hope. The grantee listed for this patent is City of Hope. Invention is credited to Yuan Chen.
United States Patent |
9,791,447 |
Chen |
October 17, 2017 |
Methods of identifying SENP1 inhibitors
Abstract
Provided herein are methods of detecting binding of an SENP1
polypeptide to a compound and methods for screening for inhibitors
of SENP1. Further provided are aqueous compositions comprising
SENP1 polypeptides and NMR apparatuses comprising the compositions
for NMR analysis.
Inventors: |
Chen; Yuan (Arcadia, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
City of Hope |
Duarte |
CA |
US |
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Assignee: |
City of Hope (Duarte,
CA)
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Family
ID: |
51654712 |
Appl.
No.: |
14/247,153 |
Filed: |
April 7, 2014 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140302525 A1 |
Oct 9, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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61809208 |
Apr 5, 2013 |
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61813832 |
Apr 19, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N
33/573 (20130101); G01N 2500/02 (20130101); G01N
2440/36 (20130101) |
Current International
Class: |
G01N
33/573 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO-2009/027973 |
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Mar 2009 |
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WO |
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WO-2009/029880 |
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Mar 2009 |
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WO |
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WO-2009/029880 |
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Mar 2009 |
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WO |
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WO-2009/029896 |
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Mar 2009 |
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WO |
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WO-2012/064887 |
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May 2012 |
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WO |
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WO 2012064898 |
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May 2012 |
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WO |
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Other References
Altschul, S.F. et al. (Sep. 1997). "Gapped BLAST and PSI-BLAST: a
new generation of protein database search programs," Nuc. Acids
Res. 25(17):3389-3402. cited by applicant .
Bang, D. et al. (Apr. 5, 2005, e-published Mar. 22, 2005). "His6
tag-assisted chemical protein synthesis," Proc. Natl. Acad. Sci.
USA, 102(14):5014-5019. cited by applicant .
Batzer, M.A. et al. (Sep. 25, 1991). "Enhanced evolutionary PCR
using oligonucleotides with inosine at the 3'-terminus," Nucleic
Acid Res. 19(18):5081. cited by applicant .
Eghbalnia, H.R. et al. (Sep. 14, 2005). High-resolution iterative
frequency identification for NMR as a general strategy for
multidimensional data collection, J. Am. Chem. Soc. 127(36):
12528-12536. cited by applicant .
Friesner, R.A. et al. (Mar. 25, 2004). "Glide: a new approach for
rapid, accurate docking and scoring. 1. Method and assessment of
docking accuracy," J Med Chem 47(7):1739-1749. cited by applicant
.
Hay, R.T. (Apr. 2005). "SUMO: a history of modification," Mol Cell
18(1):1-12. cited by applicant .
Henikoff, S. et al. (Nov. 15, 1989). "Amino acid substitution
matrices from protein blocks," Proc. Natl. Acad. Sci. USA
89(22):10915-10919. cited by applicant .
Hiller, S. et al. (Aug. 2, 2005, e-published Jul. 25, 2005).
"Automated projection spectroscopy (APSY)," Proc. Natl. Acad. Sci.
U.S.A. 102(31):10876-10881. cited by applicant .
Kolli, N. et al. (Sep. 1, 2010). "Distribution and paralogue
specificity of mammalian deSUMOylating enzymes," Biochemical
Journal 430(2):335-344. cited by applicant .
Kunkel, T.A. (Jan. 1985). "Rapid and efficient site-specific
mutagenesis without phenotypic selection," Proc. Natl. Acad. Sci.
USA 82(2):488-492. cited by applicant .
Lewis, M.K. et al. (Jun. 25, 1990). "Efficient site directed in
vitro mutagenesis using ampicillin selection," Nucl. Acids Res.,
18:3439-3443. cited by applicant .
Markley, J.L. et al. (1998). "Recommendations for the Presentation
of NMR Structures of Proteins and Nucleic Acids," Pure & Appl.
Chem. 70(1):117-142. cited by applicant .
Mikolajczyk, J. et al. (Sep. 7, 2007). "Small ubiquitin-related
modifier (SUMO)-specific proteases: profiling the specificities and
activities of human SENPs," Journal of Biological Chemistry
282(36):26217-26224. cited by applicant .
Namanja, A.T. et al. (Jan. 27, 2012, e-published Dec. 6, 2011).
"Insights into high affinity small ubiquitin-like modifier (SUMO)
recognition by SUMO-interacting motifs (SIMs) revealed by a
combination of NMR and peptide array analysis," The Journal of
Biological Chemistry 287(5):3231-3240. cited by applicant .
Nefkens, I. et al. (Feb. 1, 2003). "Heat shock and Cd2+ exposure
regulate PML and Daxx release from ND10 by independent mechanisms
that modify the induction of heat-shock proteins 70 and 25
differently," J. Cell Sci. 116(Pt. 3):513-524. cited by applicant
.
Ohtsuka, E. et al. (Mar. 10, 1985). "An alternative approach to
deoxyoligonucleotides as hybridization probes by insertion of
deoxyinosine at ambiguous codon positions," J. Biol. Chem.
260(5):2605-2608. cited by applicant .
Pearson, W.R. et al. (Apr. 1988). "Improved tools for biological
sequence comparison," Proc. Nat'l. Acad. Sci. USA 85(8):2444-2448.
cited by applicant .
Reverter, D. et al. (Dec. 2006, e-published Nov. 12, 2006).
"Structural basis for SENP2 protease interactions with SUMO
precursors and conjugated substrates," Nat. Struct. Mol. Biol.
13:1060-1068. cited by applicant .
Rossi, A.M. et al. (Mar. 2011, e-published Mar. 3, 2011). "Analysis
of protein-ligand interactions by fluorescence polarization," Nat.
Protoc. 6(3):365-387. cited by applicant .
Shen, L.N. et al. (Jul. 15, 2006). "The structure of SENP1-SUMO-2
complex suggests a structural basis for discrimination between SUMO
paralogues during processing," Biochem. J. 397(2):279-288. cited by
applicant .
Shen, L. et al. (Dec. 2006, e-published Nov. 12, 2006). "SUMO
protease SENP1 induces isomerization of the scissile peptide bond,"
Nat. Struct. Mol. Biol. 13(12):1069-1077. cited by applicant .
Shin, L. et al. (Apr. 13, 2012). "DeSUMOylating isopeptidase: a
second class of SUMO protease," EMBO Rep. 13(4):339-346. cited by
applicant .
Song, J. et al. (Oct. 5, 2004, e-published Sep. 23, 2004).
Identification of a SUMO-binding motif that recognizes
SUMO-modified proteins, PNAS USA 101(40)14373-14378. cited by
applicant .
Szyperski, T. et al. (Jun. 11, 2002). "Reduced-dimensionality NMR
spectroscopy for high-throughput protein resonance assignment,"
Proc. Natl. Acad. Sci. U.S.A. 99(12):8009-8014. cited by applicant
.
Tatham, M.H. et al. (2009). "FRET-based in vitro assays for the
analysis of SUMO protease activities," Methods Mol. Biol.
497:253-268. cited by applicant .
Xu, Z. et al. (Sep. 15, 2006). "Crystal structure of the SENP1
mutant C6035-SUMO complex reveals the hydrolytic mechanism of
SUMO-specific protease," Biochem. J. 398(3):345-352. cited by
applicant.
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Primary Examiner: Noakes; Suzanne M
Attorney, Agent or Firm: Mintz Levin Cohn Ferris Glovsky and
Popeo, P.C.
Government Interests
STATEMENT REGARDING FEDERALLY FUNDED RESEARCH
This invention was made with government support under NIH Grant
Nos. R01GM074748, R01GM086171 and R01GM102538. The government has
certain rights in the invention.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Patent
Applications 61/809,208, filed Apr. 5, 2013, and 61/813,832, filed
Apr. 19, 2013, each of which is incorporated herein by reference in
its entirety and for all purposes.
Claims
What is claimed:
1. A method of detecting binding of an SENP1 polypeptide to a
compound, the method comprising: (i) contacting an SENP1-SUMO
complex with a compound, wherein the SENP1-SUMO complex comprises
an SENP1 polypeptide and a SUMO protein; (ii) allowing the compound
to bind to the SENP1 polypeptide of the SENP1-SUMO complex, thereby
forming a SENP1-SUMO-compound complex; (iii) detecting the
SENP1-SUMO-compound complex using nuclear magnetic resonance,
thereby detecting binding of the SENP1 polypeptide to the
compound.
2. The method of claim 1, wherein the detecting comprises
determining a chemical shift for an amino acid in an active site of
the SENP1 polypeptide.
3. The method of claim 2, wherein the chemical shift in the
presence of the compound is changed relative to the corresponding
chemical shift in the absence of the compound.
4. The method of claim 3, wherein the amino acid is selected from
the group consisting of an amino acid residue corresponding to
D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469
and Q596 of SEQ ID NO: 1.
5. The method of claim 1, wherein the SENP1 polypeptide comprises
SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
6. The method of claim 1, wherein the SENP1 polypeptide comprises a
mutation at amino acid residue corresponding to amino acid residue
603 of SEQ ID NO: 1.
7. The method of claim 1, wherein the detecting comprises producing
an NMR spectra of the SENP1-SUMO-compound complex and identifying a
change in the NMR spectra relative to the absence of the
compound.
8. The method of claim 7, wherein the change is a change in the
chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.
9. The method of claim 7, wherein the change is a change in the
chemical shift of the amino acid 5603.
10. The method of claim 7, wherein the change is a change in the
chemical shift of an amino acid residue 440-455, 463-473, 493-515,
529-535, 550-554, or 596-603 of SEQ ID NO: 1.
Description
REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER PROGRAM
LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE
The Sequence Listing written in file 95058-905089_ST25.TXT, created
on Apr. 7, 2014, 22,240 bytes, machine format IBM-PC, MS-Windows
operating system, is hereby incorporated by reference.
BACKGROUND
Post-translational modifications with the small ubiquitin-like
modifiers (SUMO) are initiated and removed by the activities of
SUMO-specific proteases (SENPs). Unlike ubiquitylation, which has
one modifier (i.e., ubiquitin) and one dominant role, namely
protein degradation, SUMOylation involves three modifiers (SUMO-1,
-2, and -3) and affects diverse cellular functions. There are six
SENPs, organized into three families based on sequence similarity:
SENP1 and 2 that catalyze maturation of SUMO precursors and removal
of SUMO-1 and SUMO-2/3 conjugates; SENP3 and 5 that preferentially
remove SUMO-2/3 conjugates; and SENP6 and 7 that appear to be
mainly involved in editing poly-SUMO-2/3 chains. Recently, another
de-SUMOylase has been discovered that does not share sequence
similarity with the SENPs.
SENP inhibitors with cellular activity would be advantageous for
elucidating the role of SUMOylation in cellular regulation and for
validating SENPs as therapeutic targets. SENP1 and SENP3 are also
potential targets for developing new therapeutic agents for cancer.
They regulate the stability of hypoxia-inducible factor 1.alpha.
(HIF1.alpha.), which is a key player in the formation of new blood
vessels to support tumor growth. SENP1 is also highly expressed in
human prostate cancer specimens and regulates androgen receptor
(AR) activities. Androgen induces rapid and dynamic conjugation of
SUMO-1 to AR, while SENP1 promotes AR-dependent transcription by
cleaving SUMO-1-modified AR. SENP1 overexpression induces
transformation of normal prostate gland tissue and facilitates the
onset of high-grade prostatic intraepithelial neoplasia. Therefore,
at least some members of the SENPs are potential targets for
developing new cancer therapies.
SUMMARY
Provided herein are methods of detecting binding of an SENP1
polypeptide to a compound and methods for screening for inhibitors
of SENP1. Further provided are aqueous compositions comprising
SENP1 polypeptides and NMR apparatuses comprising the compositions
for NMR analysis.
The details of one or more embodiments are set forth in the
accompanying drawings and the description below. Other features,
objects, and advantages will be apparent from the description and
drawings, and from the claims.
DESCRIPTION OF DRAWINGS
FIG. 1 is a picture of a representative Coomassie-stained gel
showing cleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the
presence of increasing concentrations of SPI-01. YSE, fusion SUMO
(S) precursors flanked by YFP (Y) and ECFP (E) at the N- and
C-termini, respectively.
FIG. 2 is a picture of representative Coomassie-stained gel showing
cleavage of SUMO-1 and SUMO-2 by SENP1 and SENP2 in the presence of
increasing concentrations of SPI-07. YSE, fusion SUMO (S)
precursors flanked by YFP (Y) and ECFP (E) at the N- and C-termini,
respectively.
FIG. 3 is a graph showing the effects of the panel of inhibitors
shown in Table 1 at inhibiting SENP1, 2 and 7. In 96-well plates,
SENPs (50-200 nM) were pre-treated with increasing concentrations
of each compound, after which DUB-Glo (40 .mu.M final
concentration; Promega, Madison, Wis.) was added as substrate.
Experiments were performed in triplicate. The amount of cleaved
product is proportional to the relative light unit (RLU), which is
bioluminescence produced by luciferase catalyzed reaction of
luciferin that was produced by SENP cleavage of DUB-Glo.
FIG. 4 is a picture of a gel showing accumulation of
SUMO-2/3-modified proteins in HeLa cells upon treatment with
increasing doses of SPI-01.
FIG. 5 is a picture of a gel showing retention of SUMOylated
proteins during recovery of HeLa cells from heat shock in the
presence of 60 .mu.M SPI-01 and SPI-02.
FIG. 6 is a graph showing superimposition of a section of the 2D
.sup.1H-.sup.15N-heteronuclear single quantum coherence (HSQC)
spectra of the catalytically inactive C603S mutant of human SENP1
in the absence (black cross-peaks) and presence of SPI-01 (grey
cross-peaks) at 25.degree. C. Perturbed representative cross-peaks
at or near the catalytic site of SENP1 are labeled.
FIG. 7 is a graph showing the superimposition of a section of the
2D .sup.1H-.sup.15N-HSQC spectra of SUMO-1 precursor showing
labeled peaks of the C-terminal residues when free (black) and
bound to SENP1-C603S (dark grey) or both SENP1-C603S and SPI-01
(light grey) at 35.degree. C.
FIG. 8 is a picture showing all SPI-01 perturbed residues on SENP1
(PDB ID: 2IY1) labeled and colored in dark grey on the surface
representation of SENP1 in complex with SUMO-1 precursor. Perturbed
residues that are located in the vicinity of the catalytic center
of SENP1 or the C-terminus of precursor SUMO-1 are labeled in black
and grey, respectively.
FIGS. 9A and 9B are graphs showing enzyme kinetic measurements for
SPI-01 indicating a non-competitive mode of inhibition. The data
were fit to obtain the indicated kinetic parameters (.alpha.,
K.sub.i and K.sub.m) using Graphpad Prism. Lineweaver-Burk plot
analysis of the data also confirmed non-competitive inhibition.
DETAILED DESCRIPTION
SENP1 is a target for developing new therapeutic agents for cancer.
It regulates the stability of hypoxia-inducible factor 1.alpha.
(HIF1.alpha.), which is a key player in the formation of new blood
vesicles to support tumor growth. SENP1 is also highly expressed in
human prostate cancer specimens and regulates androgen receptor
(AR) activities. SENP1 is also a target for developing anti-viral
therapeutic agents for infection of viruses including, but not
limited to influenza, cytomegalovirus, herpes virus, white spot
syndrome virus, Epstein-Barr virus, adenovirus and HIV-1, because
of the role of SUMOylation in their replication. As described in
the examples below, small molecule inhibitors of SENP1 were
searched for using in-silico screening in conjunction with
biochemical assays. However, the data provided evidence for
substrate-assisted inhibitor binding. Thus, using artificial
substrates for compound screening may be misleading, as the
inhibitory effects could be significantly different from using the
physiological substrates. Therefore, embodiments are provided
including methods and inhibitors of SENP1 that confer the
non-competitive inhibitory mechanism, as shown by nuclear magnetic
resonance (NMR).
For specific SENP proteins described herein (e.g., SENP1), the
named protein includes any of the protein's naturally occurring
forms, or variants that maintain the protein activity (e.g., within
at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity
compared to the native protein). In some embodiments, variants have
at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence
identity across the whole sequence or a portion of the sequence
(e.g. a 50, 100, 150 or 200 continuous amino acid portion) compared
to a naturally occurring form. In other embodiments, the SENP1
protein is the protein as identified by its NCBI sequence
reference. In other embodiments, the SENP1 protein is the protein
as identified by its NCBI sequence reference or functional fragment
thereof.
The term "SENP1" as provided herein includes any of the
Sentrin-specific protease 1 (SENP1) naturally occurring forms,
homologs, isoforms or variants that maintain the protease activity
(e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or
100% activity compared to the native protein). In some embodiments,
variants have at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino
acid sequence identity across the whole sequence or a portion of
the sequence (e.g. a 50, 100, 150 or 200 continuous amino acid
portion) compared to a naturally occurring form. In embodiments,
the SENP1 protein is the protein as identified by the NCBI sequence
reference GI:390131988 or functional fragment thereof. In
embodiments, the SENP1 protein is the protein as identified by the
UniProt sequence reference Q9P0U3 or functional fragment thereof.
In embodiments, the SENP1 protein includes the sequence of SEQ ID
NO:1, 2, 3, 4, 5, 6, or 7. In embodiments, the SENP1 protein is
encoded by a nucleic acid sequences corresponding to Gene ID:
29843.
As described herein, nuclear magnetic resonance (NMR) approaches
have advantages over other methods previously employed on SENP1 in
identifying molecules or compounds for further development.
Specifically, the methods herein provide for discovery or
identification of compounds or inhibitors that selectively bind
SENP1 and not other SENPs. The methods also provide for
identification of compounds or inhibitors that selectively bind
SENP1-physiological substrate complexes and not SENP-artificial
substrate complexes. Further advantages include sensitivity to
binding affinities of a wide range and, thus, allowing for
identification of compounds with physicochemical properties that
are amenable for a greater scope for development of leads with
superior ADME (absorption, distribution, metabolism, and excretion)
attributes. Optionally, the test compounds are Rule-of-three (Ro3)
(MW.ltoreq.300, H-bond donors/acceptors .ltoreq.3, c Log
P.ltoreq.3, rotatable bonds .ltoreq.3) compliant (Congreve et al.,
Drug Discov. Today 8(19):876-7 (2003); and Erlanson, Top Curr.
Chem. 317:1-32 (2011)).
Nuclear magnetic resonance (NMR) studies magnetic nuclei and
provide atomic resolution information on the structures of large or
small molecules and their complexes. The elementary particles,
neutrons and protons, composing an atomic nucleus, have the
intrinsic quantum mechanical property of spin. The overall spin of
the nucleus is determined by the spin quantum number I. If the
number of both the protons and neutrons in a given isotope are
even, then I=0. In other cases, however, the overall spin is
non-zero. A non-zero spin is associated with a non-zero magnetic
moment. It is this magnetic moment that is exploited in NMR. For
example, nuclei that have a spin of one-half, like Hydrogen nuclei
(.sup.1H), a single proton, have two possible spin states (also
referred to as up and down, respectively). The energies of these
states are the same. Hence the populations of the two states (i.e.
number of atoms in the two states) will be approximately equal at
thermal equilibrium. If a nucleus is placed in a magnetic field,
however, the interaction between the nuclear magnetic moment and
the external magnetic field means the two states no longer have the
same energy. The NMR frequency (f) is shifted by the shielding
effect of the surrounding electrons. In general, this electronic
shielding reduces the magnetic field at the nucleus (which is what
determines the NMR frequency). As a result, the energy gap is
reduced, and the frequency required to achieve resonance is also
reduced. This shift of the NMR frequency due to the chemical
environment is called the chemical shift, and it explains why NMR
is a direct probe of chemical structure. The chemical shift in
absolute terms is defined by the frequency of the resonance
expressed with reference to a standard which is defined to be at 0.
The scale is made manageable by expressing it in parts per million
(ppm) of the standard frequency. Thus, in general, NMR spectral
data are reported as chemical shift and are reported in ppm
relative to either an internal standard or other baseline. A more
detailed discussion of nuclear magnetic resonance may be found in,
for example, C. P. Slichter, Principles of Magnetic Resonance, 3rd
ed., Springer-Verlag, Berlin, pp. 1-63 (1990); J. D. Roberts,
Nuclear Magnetic Resonance, Mc-Graw-Hill, N.Y., pp. 1-19 (1959);
Cohen-Tannoudji et al., Quantum Mechanics, Vol. 1, New York, N.Y.:
Wiley (1977); WO 2009/027973; WO 2009/029880; WO 2009/029896;
Hajduk et al., "High-throughput nuclear magnetic resonance-based
screening," J. Med. Chem. 42:2315-2317 (1999); and Cavanagh et al.,
Protein NMR Spectroscopy: Principles and Practice Academic Press:
San Diego (1996), which are incorporated by reference herein in
their entireties.
A variety of NMR approaches have been developed to accelerate NMR
data acquisition (Atreya et al., Methods Enzymol., 394:78-108
(2005)). For example, in the field of biological NMR spectroscopy
(Cavanagh et al., Protein NMR Spectroscopy, Academic Press: San
Diego (2007)) stable isotope (.sup.13C/.sup.15N) labeled biological
macromolecules are now studied. The isotope labeling enables one to
efficiently record three-dimensional (3D) or four-dimensional (4D)
.sup.13C/.sup.15N-resolved spectra. The most commonly used
biological NMR methods are multi-dimensional and
heteronuclear-edited NMR methods. See, for example, Tjandra and
Bax, "Direct measurement of distances and angles in biomolecules by
NMR in a dilute liquid crystalline medium," Science 1997
278(5340):1111-4 (1997). Erratum in: Science 278(5344):1697 (1997);
Clore and Gronenborn, "NMR structure determination of proteins and
protein complexes larger than 20 kDa," Curr Opin Chem. Biol.
October; 2(5):564-70 (1998); Mittermaier and Kay, "Observing
biological dynamics at atomic resolution using NMR," Trends Biochem
Sci. 34(12):601-11 (2009); and Wuthrich, Kurt, NMR of Proteins and
Nucleic Acids, John Wiley, New York, N.Y. (1986). NMR techniques
further include, but are not limited to, (i) Reduced-dimensionality
(RD) NMR (Szyperski et al., Proc. Natl. Acad. Sci. U.S.A.,
99:8009-8014 (2002)); (ii) G-matrix FT (GFT) projection NMR (Atreya
et al., J. Am. Chem. Soc., 127:4554-4555 (2005); Eletsky et al., J.
Am. Chem. Soc., 127:14578-14579 (2005); Yang et al., J. Am. Chem.
Soc., 127:9085-9099 (2005); Szyperski et al., Magn. Reson. Chem.,
44:51-60 (2006); Atreya et al., J. Am. Chem. Soc., 129:680-692
(2007); Kupce et al., J. Am. Chem. Soc., 126:6429-40 (2004); Hiller
et al., Proc. Natl. Acad. Sci. U.S.A., 102:10876-10881 (2005); and
Eghbalnia et al., J. Am. Chem. Soc., 127: 12528-12536 (2005)); and
(iii) Covariance NMR spectroscopy (Bruschweiler, J. Chem. Phys.,
121:409-414 (2004); Zhang et al., J. Am. Chem. Soc.,
126:13180-13181 (2004); and Chen et al., J. Am. Chem. Soc.,
128:15564-15565 (2006)). These publications are incorporated by
reference herein in their entireties.
Thus, as used herein, the term nuclear magnetic resonance (NMR)
encompasses a variety of methods including but not limited to,
one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR),
correlation spectroscopy NMR (COSY-NMR), total correlated
spectroscopy NMR (TOCSY-NMR), heteronuclear single-quantum
coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence
(HMQC-NMR), rotational nuclear overhauser effect spectroscopy NMR
(ROESY-NMR), nuclear overhauser effect spectroscopy (NOESY-NMR),
transverse relaxation optimized spectroscopy (TROSY-NMR) and
combinations thereof. For more description on TROSY-NMR see
Pervushin, et al., "Attenuated T.sub.2 relaxation by mutual
cancellation of dipole-dipole coupling and chemical shift
anisotropy indicates an avenue to NMR structures of very large
biological macromolecules in solution" PNAS 94:12366-71 (1997),
which is incorporated by reference herein in its entirety.
As used herein, the term "chemical shift," in nuclear magnetic
resonance (NMR) spectroscopy, refers to the resonant frequency of a
nucleus relative to a standard or baseline. Some atomic nuclei
possess a magnetic moment (nuclear spin), which gives rise to
different energy levels and resonance frequencies in a magnetic
field. The electron distribution of the same type of nucleus (e.g.
.sup.1H, .sup.13C, .sup.15N) usually varies according to the local
geometry and with it the local magnetic field at each nucleus. This
is reflected in the spin energy levels (and resonance frequencies).
The variation of nuclear magnetic resonance frequencies of the same
kind of nucleus, due to variations in the electron distribution, is
called the chemical shift. The size of the chemical shift is
typically given with respect to a reference frequency or reference
sample usually a molecule with a barely distorted electron
distribution. Typically, a .sup.1H-.sup.15N HSQC spectrum is used
to obtain chemical shift values. However, as provided in the
methods herein, any NMR analysis method can be used.
As used herein, the term "chemical shift of an amino acid" includes
the chemical shift of an element within the amino acid, e.g., H, C
or N. As used herein, the term "element" refers to an atom
distinguished by its atomic number, which is the number of protons
in its nucleus. Exemplary elements include, but are not limited to,
H (hydrogen), N (nitrogen) and C (carbon).
Exemplary chemical shift values for certain amino acids in the
SENP1 polypeptide are shown in Table 3 and exemplary chemical shift
values for certain amino acids in the SENP1 polypeptide when bound
to SUMO are shown in Table 4. The sample conditions that correlate
to the chemical shifts listed in Table 3 are 20 mM sodium
phosphate, at pH 6.8 at 25.degree. C. The sample conditions that
correlate to the chemical shifts listed in Table 4 are 20 mM sodium
phosphate and containing 150 mM NaCl, at pH 7, at 35.degree. C. The
values of the chemical shifts listed in Table 3 and Table 4 may
vary by as much as 1 ppm for .sup.1H, and as much as 5 ppm for
.sup.15N and .sup.13C due to differences in experimental conditions
such as sample pH, temperature, addition of other components (e.g.,
salt), or amino acid substitutions in SENP1 and/or SUMO that may
affect the function of SENP1 and/or SUMO. Thus, the chemical shifts
listed in Tables 3 and 4 may vary from 1 ppm for .sup.1H and from 5
ppm for .sup.15N and .sup.13C.
Thus, the peaks or chemical shifts in Tables 3 and 4 can be used by
those of skill in the art to determine whether a test compound
binds SENP1 by correlating experimental peaks or chemical shifts to
those provided in Tables 3 and 4. For example, the peaks or
chemical shifts obtained by NMR in the presence of a test compound
can be compared to the corresponding peaks or chemical shifts in
Tables 3 or 4 to determine whether the test compound binds SENP1.
Thus, the chemical shift for an amino acid of SENP1 in Table 3 or 4
can be compared to the corresponding chemical shift obtained for
the same amino acid in SENP1 in the presence of a test compound.
When performing such comparisons, one of skill in the art will
account for variances known to affect chemical shift values due to
changes in experimental conditions, e.g., pH, temperature, addition
of other components (e.g., salt), or amino acid substitutions. In
some embodiments, detection of a change of greater than 5 ppm in
the chemical shift for .sup.15N or .sup.13C of an amino acid of
SENP1 or greater than 1 ppm in the chemical shift for .sup.1H of an
amino acid of SENP1 indicates non-correlation of peaks. Optionally,
the change is as compared to the corresponding chemical shift value
for .sup.15N, .sup.13C, or .sup.1H of an amino acid of SENP1 in
Table 3 or Table 4.
As used herein, the binding of a compound to SENP1 may be
selective. The terms "selectively binds," "selectively binding," or
"specifically binding" refers to the compound binding SENP1 to the
partial or complete exclusion of other agents or compounds. By
binding is meant a detectable binding, for example, binding above
the background of the assay method. Optionally, detectable binding
is evidenced by comparing baseline to experimental values, e.g., by
comparing baseline NMR data (e.g., chemical shift values or digital
resolution spectra) to experimental NMR data (e.g., chemical shift
values or digital resolution spectra). Thus, binding can be
determined by detecting changes or perturbations in an NMR
measurement or spectrum for one sample, e.g., a control sample,
compared to another or second sample, e.g., a sample containing a
test compound. Detectable changes or perturbations in NMR signals
include changes in location (chemical shift). General NMR
techniques for proteins, including multidimensional NMR experiments
and determination of protein-ligand interactions can be found in
David G. Reid (ed.), Protein NMR Techniques, Humana Press, Totowa
N.J. (1997). By way of example, detection of a perturbation or
change includes detection of a difference in the chemical shift of
SENP1 or SENP1-SUMO complex in the presence of a compound as
compared to the chemical shift in the absence of the compound. The
perturbation or change (whether increased or decreased) can include
significant differences in an NMR measurement or spectrum (e.g.,
chemical shift) and can be greater than the experimental error or
greater than the error bar range. For example, a change of at least
about 1.1 times of the digital resolution of a spectrum or chemical
shift for one or more amino acid residues of SENP1 in the presence
of a compound can indicate the compound binds SENP1. Thus, a change
of at least about 1.1, 1.2, 1.3, 1.4, 1.5, 2, 2.5, 3, 3.5, 4, 4.5,
5, 10, 20 times or more of the digital resolution of an NMR
measurement or spectrum, e.g., chemical shift, observed in the
presence of a compound as compared to a control can indicate the
compound binds SENP1.
The terms greater, higher, increases, elevates, or elevation refer
to increases above a control. The terms low, lower, reduces, or
reduction refer to any decrease below control levels. For example,
control levels are levels prior to, or in the absence of, addition
of a compound.
A "control" sample or value refers to a sample that serves as a
reference, usually a known reference, for comparison to a test
sample. For example, a test sample can be taken from a test
condition, e.g., in the presence of a test compound, and compared
to samples from known conditions, e.g., in the absence of the test
compound (negative control), or in the presence of a known compound
(positive control). A control can also represent an average value
gathered from a number of tests or results. One of skill in the art
will recognize that controls can be designed for assessment of any
number of parameters. For example, a control can be devised to
compare therapeutic benefit based on pharmacological data (e.g.,
half-life) or therapeutic measures (e.g., comparison of side
effects). One of skill in the art will understand which controls
are valuable in a given situation and be able to analyze data based
on comparisons to control values. Controls are also valuable for
determining the significance of data. For example, if values for a
given parameter are widely variant in controls, variation in test
samples will not be considered as significant.
"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides
and polymers thereof in either single- or double-stranded form, and
complements thereof. The term encompasses nucleic acids containing
known nucleotide analogs or modified backbone residues or linkages,
which are synthetic, naturally occurring, and non-naturally
occurring, which have similar binding properties as the reference
nucleic acid, and which are metabolized in a manner similar to the
reference nucleotides. Examples of such analogs include, without
limitation, phosphorothioates, phosphoramidates, methyl
phosphonates, chiral-methyl phosphonates, 2-O-methyl
ribonucleotides, peptide-nucleic acids (PNAs).
Unless otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. Specifically,
degenerate codon substitutions may be achieved by generating
sequences in which the third position of one or more selected (or
all) codons is substituted with mixed-base and/or deoxyinosine
residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka
et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol.
Cell. Probes 8:91-98 (1994)). The term nucleic acid is used
interchangeably with gene, cDNA, mRNA, oligonucleotide, and
polynucleotide.
"Percentage of sequence identity" is determined by comparing two
optimally aligned sequences over a comparison window, wherein the
portion of the polynucleotide or polypeptide sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
The percentage is calculated by determining the number of positions
at which the identical nucleic acid base or amino acid residue
occurs in both sequences to yield the number of matched positions,
dividing the number of matched positions by the total number of
positions in the window of comparison and multiplying the result by
100 to yield the percentage of sequence identity.
The terms "identical" or percent "identity," in the context of two
or more nucleic acids or polypeptide sequences, refer to two or
more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identity over a
specified region, e.g., of the entire polypeptide sequences of the
invention or individual domains of the polypeptides of the
invention), when compared and aligned for maximum correspondence
over a comparison window, or designated region as measured using
one of the following sequence comparison algorithms or by manual
alignment and visual inspection. Such sequences are then said to be
"substantially identical." This definition also refers to the
complement of a test sequence. Optionally, the identity exists over
a region that is at least about 10 to about 100, about 20 to about
75, about 30 to about 50 amino acids or nucleotides in length.
Optionally, the identity exists over a region that is at least
about 50 amino acids in length, or more preferably over a region
that is 100 to 500 or 1000 or more amino acids in length. The
present invention includes polypeptides that are substantially
identical to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
For sequence comparison, typically one sequence acts as a reference
sequence, to which test sequences are compared. When using a
sequence comparison algorithm, test and reference sequences are
entered into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. Preferably, default program parameters can be used, or
alternative parameters can be designated. The sequence comparison
algorithm then calculates the percent sequence identities for the
test sequences relative to the reference sequence, based on the
program parameters.
A "comparison window", as used herein, includes reference to a
segment of any one of the number of contiguous positions selected
from the group consisting of from 20 to 600, usually about 50 to
about 200, more usually about 100 to about 150 in which a sequence
may be compared to a reference sequence of the same number of
contiguous positions after the two sequences are optimally aligned.
Methods of alignment of sequences for comparison are well-known in
the art. Optimal alignment of sequences for comparison can be
conducted, e.g., by the local homology algorithm of Smith &
Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment
algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),
by the search for similarity method of Pearson & Lipman, Proc.
Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package, Genetics
Computer Group, 575 Science Dr., Madison, Wis.), or by manual
alignment and visual inspection (see, e.g., Current Protocols in
Molecular Biology (Ausubel et al., eds. 1995 supplement)).
A preferred example of algorithm that is suitable for determining
percent sequence identity and sequence similarity are the BLAST and
BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.
215:403-410 (1990), respectively. BLAST and BLAST 2.0 are used,
with the parameters described herein, to determine percent sequence
identity for the nucleic acids and proteins. Software for
performing BLAST analyses is publicly available through the
National Center for Biotechnology Information, as known in the art.
This algorithm involves first identifying high scoring sequence
pairs (HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always >0) and N (penalty score for
mismatching residues; always <0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, M=5, N=-4 and a comparison of both strands. For amino
acid sequences, the BLASTP program uses as defaults a wordlength of
3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see
Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915
(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and
a comparison of both strands.
The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical mimetic of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers and non-naturally occurring
amino acid polymer.
The term "amino acid" refers to naturally occurring and synthetic
amino acids, as well as amino acid analogs and amino acid mimetics
that function in a manner similar to the naturally occurring amino
acids. Naturally occurring amino acids are those encoded by the
genetic code, as well as those amino acids that are later modified,
e.g., hydroxyproline, .gamma.-carboxyglutamate, and
O-phosphoserine. Amino acid analogs refers to compounds that have
the same basic chemical structure as a naturally occurring amino
acid, i.e., an .alpha. carbon that is bound to a hydrogen, a
carboxyl group, an amino group, and an R group, e.g., homoserine,
norleucine, methionine sulfoxide, methionine methyl sulfonium. Such
analogs have modified R groups (e.g., norleucine) or modified
peptide backbones, but retain the same basic chemical structure as
a naturally occurring amino acid. Amino acid mimetics refers to
chemical compounds that have a structure that is different from the
general chemical structure of an amino acid, but that functions in
a manner similar to a naturally occurring amino acid.
Amino acids may be referred to herein by either their commonly
known three letter symbols or by the one-letter symbols recommended
by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides,
likewise, may be referred to by their commonly accepted
single-letter codes.
An amino acid residue in a polypeptide "corresponds" to or "is
corresponding to" a given residue when it occupies the same
essential structural position within the polypeptide as the given
residue. For example, a selected residue in a comparison
polypeptide corresponds to position 603 in a polypeptide provided
herein (e.g., a SENP1 polypeptide), when the selected residue
occupies the same essential spatial or structural relationship to
position 603 as assessed using applicable methods in the art. For
example, a comparison polypeptide may be aligned for maximum
sequence homology with the polypeptide provided herein and the
position in the aligned comparison polypeptide that aligns with
position 603 may be determined to correspond to it. Alternatively,
instead of (or in addition to) a primary sequence alignment as
described above, a three dimensional structural alignment can also
be used, e.g., where the structure of the comparison polypeptide is
aligned for maximum correspondence with a polypeptide provided
herein and the overall structures compared. In this case, an amino
acid that occupies the same essential position as position 603 in
the structural model may be said to correspond.
"Conservatively modified variants" applies to both amino acid and
nucleic acid sequences. With respect to particular nucleic acid
sequences, conservatively modified variants refers to those nucleic
acids which encode identical or essentially identical amino acid
sequences, or where the nucleic acid does not encode an amino acid
sequence, to essentially identical sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
conservatively modified variations. Every nucleic acid sequence
herein which encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of skill will recognize
that each codon in a nucleic acid (except AUG, which is ordinarily
the only codon for methionine, and TGG, which is ordinarily the
only codon for tryptophan) can be modified to yield a functionally
identical molecule. Accordingly, each silent variation of a nucleic
acid which encodes a polypeptide is implicit in each described
sequence with respect to the expression product, but not with
respect to actual probe sequences.
As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" where
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. Such conservatively modified variants are in addition to and
do not exclude polymorphic variants, interspecies homologs, and
alleles.
The following eight groups each contain amino acids that are
conservative substitutions for one another: 1) Alanine (A), Glycine
(G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N),
Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I),
Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F),
Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8)
Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins
(1984)).
The "active-site" of a protein or polypeptide refers to a protein
domain that is structurally, functionally, or both structurally and
functionally, active. For example, the active-site of a protein can
be a site that catalyzes an enzymatic reaction, i.e., a
catalytically active site. An active site refers to a domain that
includes amino acid residues involved in binding of a substrate for
the purpose of facilitating the enzymatic reaction. Optionally, the
term active site refers to a protein domain that binds to another
agent, molecule or polypeptide. For example, the active sites of
SENP1 include sites on SENP1 that bind to or interact with SUMO. A
protein may have one or more active-sites.
The phrase "stringent hybridization conditions" refers to
conditions under which a probe will hybridize to its target
subsequence, typically in a complex mixture of nucleic acids, but
to no other sequences. Stringent conditions are sequence-dependent
and will be different in different circumstances. Longer sequences
hybridize specifically at higher temperatures. An extensive guide
to the hybridization of nucleic acids is found in Tijssen,
Techniques in Biochemistry and Molecular Biology--Hybridization
with Nucleic Probes, "Overview of principles of hybridization and
the strategy of nucleic acid assays" (1993). Generally, stringent
conditions are selected to be about 5-10.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength pH. The T.sub.m is the temperature (under
defined ionic strength, pH, and nucleic concentration) at which 50%
of the probes complementary to the target hybridize to the target
sequence at equilibrium (as the target sequences are present in
excess, at T.sub.m, 50% of the probes are occupied at equilibrium).
Stringent conditions may also be achieved with the addition of
destabilizing agents such as formamide. For selective or specific
hybridization, a positive signal is at least two times background,
preferably 10 times background hybridization. Exemplary stringent
hybridization conditions can be as following: 50% formamide,
5.times.SSC, and 1% SDS, incubating at 42.degree. C., or,
5.times.SSC, 1% SDS, incubating at 65.degree. C., with wash in
0.2.times.SSC, and 0.1% SDS at 65.degree. C.
Nucleic acids that do not hybridize to each other under stringent
conditions are still substantially identical if the polypeptides
which they encode are substantially identical. This occurs, for
example, when a copy of a nucleic acid is created using the maximum
codon degeneracy permitted by the genetic code. In such cases, the
nucleic acids typically hybridize under moderately stringent
hybridization conditions. Exemplary "moderately stringent
hybridization conditions" include a hybridization in a buffer of
40% formamide, 1 M NaCl, 1% SDS at 37.degree. C., and a wash in
1.times.SSC at 45.degree. C. A positive hybridization is at least
twice background. Those of ordinary skill will readily recognize
that alternative hybridization and wash conditions can be utilized
to provide conditions of similar stringency. Additional guidelines
for determining hybridization parameters are provided in numerous
reference, e.g., and Current Protocols in Molecular Biology, ed.
Ausubel, et al., John Wiley & Sons.
For PCR, a temperature of about 36.degree. C. is typical for low
stringency amplification, although annealing temperatures may vary
between about 32.degree. C. and 48.degree. C. depending on primer
length. For high stringency PCR amplification, a temperature of
about 62.degree. C. is typical, although high stringency annealing
temperatures can range from about 50.degree. C. to about 65.degree.
C., depending on the primer length and specificity. Typical cycle
conditions for both high and low stringency amplifications include
a denaturation phase of 90.degree. C.-95.degree. C. for 30 sec-2
min., an annealing phase lasting 30 sec.-2 min., and an extension
phase of about 72.degree. C. for 1-2 min. Protocols and guidelines
for low and high stringency amplification reactions are provided,
e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and
Applications, Academic Press, Inc. N.Y.).
The term "pharmaceutically acceptable salts" or "pharmaceutically
acceptable carrier" is meant to include salts of the active
compounds which are prepared with relatively nontoxic acids or
bases, depending on the particular substituents found on the
compounds described herein. When compounds of the present
application contain relatively acidic functionalities, base
addition salts can be obtained by contacting the neutral form of
such compounds with a sufficient amount of the desired base, either
neat or in a suitable inert solvent. Examples of pharmaceutically
acceptable base addition salts include sodium, potassium, calcium,
ammonium, organic amino, or magnesium salt, or a similar salt. When
compounds of the present application contain relatively basic
functionalities, acid addition salts can be obtained by contacting
the neutral form of such compounds with a sufficient amount of the
desired acid, either neat or in a suitable inert solvent. Examples
of pharmaceutically acceptable acid addition salts include those
derived from inorganic acids like hydrochloric, hydrobromic,
nitric, carbonic, monohydrogencarbonic, phosphoric,
monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,
monohydrogensulfuric, hydriodic, or phosphorous acids and the like,
as well as the salts derived from relatively nontoxic organic acids
like acetic, propionic, isobutyric, maleic, malonic, benzoic,
succinic, suberic, fumaric, lactic, mandelic, phthalic,
benzenesulfonic, p-tolylsulfonic, citric, tartaric,
methanesulfonic, and the like. Also included are salts of amino
acids such as arginate and the like, and salts of organic acids
like glucuronic or galactunoric acids and the like (see, e.g.,
Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)).
Other pharmaceutically acceptable carriers known to those of skill
in the art are suitable for compositions of the present
application.
A "label" or a "detectable moiety" is a composition detectable by
spectroscopic, photochemical, biochemical, immunochemical,
chemical, or other physical means. For example, useful labels
include .sup.32P, fluorescent dyes, electron-dense reagents,
enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin,
or haptens and proteins or other entities which can be made
detectable, e.g., by incorporating a fluorescent label into a
peptide specifically reactive with a target peptide (e.g., SENP1
polypeptide, SUMO protein or test compound). In embodiments, the
label is a fluorescent label. Any method known in the art for
conjugating a polypeptide to the label may be employed, e.g., using
methods described in Hermanson, Bioconjugate Techniques 1996,
Academic Press, Inc., San Diego.
A "labeled protein or polypeptide" is one that is bound, either
covalently, through a linker or a chemical bond, or noncovalently,
through ionic, van der Waals, electrostatic, or hydrogen bonds to a
label such that the presence of the labeled protein or polypeptide
may be detected by detecting the presence of the label bound to the
labeled protein or polypeptide.
Methods
Provided herein are methods of detecting binding of an SENP1
polypeptide to a compound. The method includes the steps of
contacting an SENP1 polypeptide with a compound, allowing the
compound to bind to the SENP1 polypeptide, thereby forming a
SENP1-compound complex, and detecting the SENP1-compound complex
using nuclear magnetic resonance, thereby detecting binding of the
SENP1 polypeptide to the compound.
A "compound" as provided herein refers to a polypeptide, protein,
amino acid, small molecule or chemical compound that is capable of
binding a SENP1 polypeptide or fragment thereof. In embodiments,
the compound binds a SENP1 protein of SEQ ID NO:1, 2, 3, 4, 5, 6,
or 7. In embodiments, the compound is a modulator of SENP1
activity. In embodiments, the compound is an inhibitor of SENP1
activity. In embodiments, the compound is an activator of SENP1
activity. In embodiments, the compound is a small molecule. A small
molecule as provided herein include, but are not limited to the
compounds in Tables 1 and 2 and those described in WO 2012/064887,
which is incorporated by reference herein in its entirety. As used
herein, the term "small molecule" refers to an organic compound
containing carbon. A small molecule is generally, but not
necessarily, of low molecular weight, e.g., less than 1000
Daltons.
A "test compound" as provided herein refers to a compound useful
for the screening methods provided herein. A test compound may be
capable of binding a SENP1 polypeptide or fragment thereof as
provided herein. In embodiments, the test compound binds a SENP1
polypeptide or fragment thereof. In embodiments, the binding of the
test compound to the SENP1 polypeptide or fragment thereof is
detected by nuclear magnetic resonance. In embodiments, the test
compound does not bind a SENP1 polypeptide or fragment thereof.
As defined herein, the term "inhibition", "inhibit", "inhibiting"
and the like in reference to a compound or protein-inhibitor
interaction means negatively affecting (e.g., decreasing) the
activity or function of the protein (e.g. decreasing gene
transcription or translation) relative to the activity or function
of the protein in the absence of the inhibitor. In embodiments,
inhibition refers to reduction of a disease or symptoms of disease
(e.g., cancer). In embodiments, inhibition refers to a reduction in
the activity of an enzymatic activity (e.g., SENP activity). In
embodiments, inhibition refers to a reduction in the activity of a
signal transduction pathway or signaling pathway (e.g. cell cycle).
Thus, inhibition includes, at least in part, partially or totally
blocking stimulation, decreasing, preventing, or delaying
activation, or inactivating, desensitizing, or down-regulating
transcription, translation, signal transduction or enzymatic
activity or the amount of a protein (e.g. a cellular protein or a
viral protein). In embodiments, inhibition refers to inhibition of
SENP1.
The terms "inhibitor," "repressor" or "antagonist" or
"downregulator" interchangeably refer to a substance that results
in a detectably lower expression or activity level as compared to a
control. The inhibited expression or activity can be 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90% or less than that in a control. In
certain instances, the inhibition is 1.5-fold, 2-fold, 3-fold,
4-fold, 5-fold, 10-fold, or more in comparison to a control. An
"inhibitor" is a siRNA, (e.g., shRNA, miRNA, snoRNA), compound or
small molecule that inhibits cellular function (e.g., replication)
e.g., by binding, partially or totally blocking stimulation,
decrease, prevent, or delay activation, or inactivate, desensitize,
or down-regulate signal transduction, gene expression or enzymatic
activity necessary for protein activity Inhibition as provided
herein may also include decreasing or blocking a protein activity
(e.g., activity of SENP1).
The terms "agonist," "activator," "upregulator," etc. refer to a
substance capable of detectably increasing the expression or
activity of a given gene or protein. The agonist can increase
expression or activity 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%
or more in comparison to a control in the absence of the agonist.
In certain instances, expression or activity is 1.5-fold, 2-fold,
3-fold, 4-fold, 5-fold, 10-fold or higher than the expression or
activity in the absence of the agonist.
Optionally, the compound is a small molecule. Optionally, the step
of detecting includes detecting a perturbation in the presence of
the compound relative to the absence of the compound. For example,
binding of a compound to SENP1 is detected if a perturbation is
detected in an NMR measurement or spectrum in the presence of the
compound as compared to or relative to the absence of the compound.
Optionally, the step of detecting includes determining a chemical
shift for an amino acid in an active site of the SENP1 polypeptide.
Binding is detected by a change in the chemical shift in the
presence of the compound relative to the corresponding chemical
shift in the absence of the compound. Optionally, the active site
is a catalytically active site. Optionally, the active site is a
site involved in SUMO binding, e.g., the active site is a site on
SENP1 that binds to the SUMO protein. Thus, the step of detecting
includes determining a chemical shift for an amino acid involved in
binding of SENP1 polypeptide to SUMO. Optionally, the chemical
shift is determined for one or more amino acids of SEQ ID NOs: 3,
4, 5, 6 or 7.
Optionally, the chemical shift is determined for one or more amino
acid residues selected from the group consisting of D550, H533,
C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of
SEQ ID NO:1.
In embodiments, the change is a change in the chemical shift of
amino acid residue D550, H533, C603, W465, W534, L466, G531, C535,
M552, G554, E469 or Q596 of SEQ ID NO:1. In embodiments, the change
is a change in the chemical shift of amino acid residue D550, H533,
C603, W465, W534, L466, G531, C535, M552, G554, E469 and Q596 of
SEQ ID NO:1. In embodiments, the change is a change in the chemical
shift of amino acid residue D550 of SEQ ID NO:1. In embodiments,
the change is a change in the chemical shift of amino acid residue
H533 of SEQ ID NO:1. In embodiments, the change is a change in the
chemical shift of amino acid residue C603 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of amino
acid residue W465 of SEQ ID NO:1. In embodiments, the change is a
change in the chemical shift of amino acid residue W534 of SEQ ID
NO:1. In embodiments, the change is a change in the chemical shift
of amino acid residue L466 of SEQ ID NO:1. In embodiments, the
change is a change in the chemical shift of amino acid residue G531
of SEQ ID NO:1. In embodiments, the change is a change in the
chemical shift of amino acid residue C535 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of amino
acid residue M552 of SEQ ID NO:1. In embodiments, the change is a
change in the chemical shift of amino acid residue G554 of SEQ ID
NO:1. In embodiments, the change is a change in the chemical shift
of amino acid residue E469 of SEQ ID NO:1. In embodiments, the
change is a change in the chemical shift of amino acid residue Q596
of SEQ ID NO:1.
In embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to D550, H533, C603, W465, W534,
L466, G531, C535, M552, G554, E469 or Q596 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to D550, H533, C603, W465, W534,
L466, G531, C535, M552, G554, E469 and Q596 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to D550 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to H533 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to C603 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to W465 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to W534 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to L466 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to G531 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to C535 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to M552 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to G554 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to E469 of SEQ ID NO:1. In
embodiments, the change is a change in the chemical shift of an
amino acid residue corresponding to Q596 of SEQ ID NO:1.
In embodiments, the SENP1 polypeptide includes amino acid residue
603 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes
an amino acid residue corresponding to amino acid residue 603 of
SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes amino
acid residue 550 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes an amino acid residue corresponding to amino
acid residue 550 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes amino acid residue 533 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes an amino acid residue
corresponding to amino acid residue 533 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes amino acid residue 465
of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an
amino acid residue corresponding to amino acid residue 465 of SEQ
ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid
residue 534 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide
includes an amino acid residue corresponding to amino acid residue
534 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes
amino acid residue 466 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes an amino acid residue corresponding to amino
acid residue 466 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes amino acid residue 531 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes an amino acid residue
corresponding to amino acid residue 531 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes amino acid residue 535
of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an
amino acid residue corresponding to amino acid residue 535 of SEQ
ID NO:1. In embodiments, the SENP1 polypeptide includes amino acid
residue 552 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide
includes an amino acid residue corresponding to amino acid residue
552 of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes
amino acid residue 554 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes an amino acid residue corresponding to amino
acid residue 554 of SEQ ID NO:1. In embodiments, the SENP1
polypeptide includes amino acid residue 469 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes an amino acid residue
corresponding to amino acid residue 469 of SEQ ID NO:1. In
embodiments, the SENP1 polypeptide includes amino acid residue 596
of SEQ ID NO:1. In embodiments, the SENP1 polypeptide includes an
amino acid residue corresponding to amino acid residue 596 of SEQ
ID NO:1.
Optionally, the chemical shift is determined for a mutation at
amino acid residue 603 of SEQ ID NO:1. Optionally, the mutation is
C603S. Optionally, the chemical shift is determined for one or more
amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or
596-603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide or
SENP1-compound complex is bound to a SUMO protein thereby forming a
SENP1-SUMO complex or SENP1-SUMO-compound complex. Optionally, the
SUMO protein is a truncated SUMO protein. Optionally, the compound
does not interact with C603 of SEQ ID NO:1 of SENP1, e.g., the
compound does not covalently modify C603 of SENP1. Thus, the
provided methods optionally include detecting binding by producing
an NMR spectra of the SENP-1 compound complex and identifying a
change in the NMR spectra relative to the absence of the compound.
Optionally, the change is a change in the chemical shift of an
amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7. Optionally, the change
is a change in the chemical shift of an amino acid selected from
the group consisting of D550, H533, C603, W465, W534, L466, G531,
C535, M552, G554, E469 and Q596. Optionally, the change is a change
in the chemical shift of the amino acid 5603. Optionally, the
change is a change in the chemical shift of an amino acid residue
440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID
NO:1.
Also provided is a method of screening for compounds that bind
SENP1 including the steps of providing a first sample comprising
SENP1 or an SENP1-SUMO complex, determining an NMR spectra of the
first sample, providing a second sample comprising an
SENP1-compound complex or an SENP1-SUMO-compound complex, and
determining an NMR spectra of the second sample. Detection of a
change in the NMR spectra in the second sample as compared to the
first sample indicates the compound binds SENP1.
Provided are methods of screening for an inhibitor of SENP1. The
methods include contacting a composition comprising an SENP1
polypeptide with a test compound and detecting whether the test
compound binds the SENP1 polypeptide or fragment thereof by nuclear
magnetic resonance.
Optionally, the step of detecting includes detecting a perturbation
in the presence of the compound relative to the absence of the
compound. For example, the test compound binds or inhibits SENP1 if
a perturbation is detected in an NMR measurement or spectrum in the
presence of the compound as compared to or relative to the absence
of the compound. Optionally, the step of detecting comprises
determining a chemical shift for one or more amino acids in the
active site of the SENP1 polypeptide. The chemical shift in the
presence of the compound will be changed relative to the
corresponding chemical shift in the absence of the test compound if
the test compound binds to SENP1. Optionally, the active site is a
catalytically active site. Optionally, the active site is a site
involved in SUMO binding, e.g., the active site is a site on SENP1
that binds to the SUMO protein. Thus, the step of detecting
includes determining a chemical shift for an amino acid involved in
binding of SENP1 polypeptide to SUMO. Optionally, the chemical
shift is determined for one or more amino acids of SEQ ID NOs: 3,
4, 5, 6 OR 7. Optionally, the chemical shift is determined for one
or more amino acid residues selected from the group consisting of
D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469
and Q596 of SEQ ID NO:1. Optionally, the chemical shift is
determined for a mutation at amino acid residue 603 of SEQ ID NO:1.
Optionally, the mutation is C603S. Optionally, the chemical shift
is determined for one or more amino acid residues 440-455, 463-473,
493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally,
the SENP1 polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO complex. Optionally, the SUMO protein is a truncated
SUMO protein. Optionally, the composition comprising the SENP1
polypeptide or SENP1-SUMO complex is an aqueous solution.
Optionally, the composition is at a pH from about 6.0 to about 7.5.
Optionally, the pH is about 6.8. Optionally, the composition
comprises a buffering agent, a reducing agent, a base or
combinations thereof. Optionally, the composition comprises sodium
phosphate, D.sub.2O, sodium azide, dithiothreitol or combinations
thereof. The sodium phosphate can be present at about 20 mM.
Optionally, the compound to be tested is a small molecule.
Optionally, the compound does not interact with C603 numbered
relative to SEQ ID NO:1 of SENP1, e.g., the compound does not
covalently modify C603 of SENP1. Optionally, in the provided
methods, the SENP1 binds the compound forming an SENP1-compound
complex and the detecting comprises producing an NMR spectra of the
SENP1-compound complex and identifying a change in the NMR spectra
relative to the absence of the compound. Optionally, the change is
a change in the chemical shift of an amino acid of SEQ ID NOs: 3,
4, 5, 6 or 7. Optionally, the change is a change in the chemical
shift of an amino acid selected from the group consisting of D550,
H533, C603, W465, W534, L466, G531, C535, M552, G554, E469 and
Q596. Optionally, the change is a change in the chemical shift of
the amino acid 5603. Optionally, the change is a change in the
chemical shift of an amino acid residue 440-455, 463-473, 493-515,
529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally, the change
is a change in the chemical shift of an amino acid in the active
site of SENP1. Optionally, the active site is a catalytically
active site or a site that binds to the SUMO protein.
Also provided are methods of identifying an SENP1 inhibitor that
include combining an SENP1 polypeptide, a SUMO protein, and a test
compound in a reaction vessel, allowing the SENP1 polypeptide, SUMO
protein and test compound to form a SENP1-SUMO-compound complex,
and detecting the SENP1-SUMO-compound complex thereby identifying
the compound as a SENP1 inhibitor. A "reaction vessel" as provided
herein refers to a vial, tube, flask, bottle, syringe or other
container means, into which the SENP1 polypeptide, SUMO protein and
test compound are combined to allow the formation of a
SENP1-SUMO-compound complex.
Optionally, one or more of the SENP1 polypeptide, SUMO protein or
test compound is labeled. Optionally, the label is a fluorescent
label. Optionally, the test compound comprises a fluorescent label.
Optionally, the SUMO is a truncated SUMO protein. Optionally, the
SUMO comprises amino acid residues 1-92 of the SUMO protein.
Optionally, the SUMO protein comprises SEQ ID NO:8 or SEQ ID NO:9.
Optionally, the SENP1 polypeptide comprises SEQ ID NOs: 1, 2, 3, 4,
5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid
residue 603 of SEQ ID NO:1. Optionally, the SENP1 polypeptide
comprises a mutation at amino acid residue 603 of SEQ ID NO:1.
Optionally, the mutation is C603S. Optionally, the SENP1
polypeptide comprises amino acid residues 440-455, 463-473,
493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1. Optionally,
the test compound is a small molecule. In the provided methods, the
detecting can be performed by a variety of methods known to those
skilled in the art and described in the example below. See, e.g.,
Protein-Ligand Interactions, Vol. 1008, Methods in Molecular
Biology, Humana Press, Inc., Clifton, N.J., Williams and Daviter,
Eds. (2013). For example, a wide variety of assays for detecting
binding can be used including labeled in vitro protein-ligand
binding assays, cell based assays, immunoassays, and the like.
Optionally, detecting can be performed using solution-phase binding
assays, e.g., fluorescent polarization. Thus, binding can be
detected by fluorescent polarization (Rossi et al., Nat. Protoc.
6(3):365-87 (2011)). Optionally, binding is detected by detecting a
change in the thermal properties of SENP1, e.g., the thermal
property can be the melting temperature of SENP1. In some
embodiments, the detecting is performed using nuclear magnetic
resonance. Optionally, the detecting comprises producing an NMR
spectra of the SENP1-SUMO-compound complex and identifying a change
in the NMR spectra relative to the absence of the test compound.
Optionally, the change is a change in the chemical shift of an
amino acid in an active site of the SENP1 polypeptide. The active
site can be, for example, a catalytically active site or a site
that binds to the SUMO protein. Optionally, the amino acid is an
amino acid of SEQ ID NOs: 3, 4, 5, 6 OR 7. Optionally, the amino
acid is selected from the group consisting of D550, H533, C603,
W465, W534, L466, G531, C535, M552, G554, E469 and Q596.
Optionally, the amino acid is 5603. Optionally, the amino acid is
amino acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or
596-603 of SEQ ID NO:1.
As used throughout, the term "SENP1 polypeptide" refers to full
length SENP1 and fragments thereof. The sequence and structure of
the SENP1 polypeptide is known. (See above and Protein Data Bank
(PDB) accession codes 2IYC and 2IY1; Shen et al., Nat. Struct. Mol.
Biol. 13(12):1069-1077 (2006); and Xu et al., Biochem. J.
398(3):345-52 (2006)). Optionally, the SENP1 polypeptide comprises
SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1
polypeptide comprises amino acid residue 603 of SEQ ID NO:1.
Optionally, the SENP1 polypeptide comprises a mutation at amino
acid residue 603 of SEQ ID NO:1. Optionally, the mutation is C603S.
Optionally, the SENP1 polypeptide comprises amino acid residues
440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of SEQ ID
NO:1.
Optionally, in the provided methods, SENP1 is bound to SUMO or a
fragment thereof, e.g., a truncated SUMO protein. Thus, optionally,
the SENP1 is bound to a SUMO protein thereby forming a SENP1-SUMO
complex. Optionally, the SUMO protein is a truncated SUMO protein.
Optionally, the SUMO protein is SEQ ID NO:8 or 9. As used herein,
the term "truncated SUMO protein" refers to a SUMO protein or
polypeptide that has been manipulated to remove at least one amino
acid residue relative to wild-type SUMO, e.g., a SUMO protein or
polypeptide that occurs in nature. Exemplary wild-type SUMO
proteins include, but are not limited to, SEQ ID NO:9 and those
found at GenBank Accession Nos. AAC50996.1, NP_008868.3,
NP_001005849.1, P55854.2, and NP_008867.2. Truncated SUMO proteins
include, but are not limited to, SEQ ID NO:8. As used herein, the
term "SUMO" refers to SUMO1, SUMO2, or SUMO3 or fragments thereof
or complexes thereof, e.g., SUMO2/3. The nucleic acid and amino
acid sequences for SUMO are known. See, for example, Hay, Mol.
Cell. 18(1):1-12 (2005); and Yeh, et al., J. Biol. Chem.,
284(13):8223-7 (2009). For example, nucleic acid and amino acid
sequences for SUMO-1 can be found at GenBank Accession Nos.
U67122.1 and AAC50996.1. Nucleic acid and amino acid sequences for
SUMO-2 can be found at GenBank Accession Nos. NM_006937.3,
NM_001005849.1, NP_008868.3 and NP_001005849.1. Nucleic acid and
amino acid sequences for SUMO-3 can be found at GenBank Accession
Nos. NM_006936.2, P55854.2, and NP_008867.2. Optionally, the SENP1
is bound to SUMO1 to form an SENP1-SUMO1 complex.
The provided SENP1 polypeptides and/or SUMO polypeptides and
fragments thereof may contain one or more modifications, e.g., a
conservative modification. As used herein, the term "modification"
refers to a modification in a nucleic acid sequence of a gene or an
amino acid sequence. Modifications include, but are not limited to,
insertions, substitutions and deletions. Amino acid sequence
modifications typically fall into one or more of three classes:
substitutional, insertional, or deletional modifications.
Insertions include amino and/or terminal fusions as well as
intrasequence insertions of single or multiple amino acid residues.
Insertions ordinarily will be smaller insertions than those of
amino or carboxyl terminal fusions, for example, on the order of
one to four residues. Deletions are characterized by the removal of
one or more amino acid residues from the protein sequence. Amino
acid substitutions are typically of single residues, but can occur
at a number of different locations at once. Substitutions,
deletions, insertions or any combination thereof may be combined to
arrive at a final construct. Substitutional modifications are those
in which at least one residue has been removed and a different
residue inserted in its place.
Modifications are generated using any number of methods known in
the art. For example, site directed mutagenesis can be used to
modify a nucleic acid sequence. One of the most common methods of
site-directed mutagenesis is oligonucleotide-directed mutagenesis.
In oligonucleotide-directed mutagenesis, an oligonucleotide
encoding the desired change(s) in sequence is annealed to one
strand of the DNA of interest and serves as a primer for initiation
of DNA synthesis. In this manner, the oligonucleotide containing
the sequence change is incorporated into the newly synthesized
strand. See, for example, Kunkel, 1985, Proc. Natl. Acad. Sci. USA,
82:488; Kunkel et al., 1987, Meth. Enzymol., 154:367; Lewis &
Thompson, 1990, Nucl. Acids Res., 18:3439; Bohnsack, 1996, Meth.
Mol. Biol., 57:1; Deng & Nickoloff, 1992, Anal. Biochem.,
200:81; and Shimada, 1996, Meth. Mol. Biol., 57:157. Other methods
are routinely used in the art to introduce a modification into a
sequence. For example, modified nucleic acids are generated using
PCR or chemical synthesis, or polypeptides having the desired
change in amino acid sequence can be chemically synthesized. See,
for example, Bang & Kent, 2005, Proc. Natl. Acad. Sci. USA,
102:5014-9 and references therein.
Also provided herein are nucleic acids encoding the polypeptides
described throughout. It is understood that the nucleic acids that
can encode those peptide, polypeptide, or protein sequences,
variants and fragments thereof are also disclosed. This would
include all degenerate sequences related to a specific polypeptide
sequence, i.e. all nucleic acids having a sequence that encodes one
particular polypeptide sequence as well as all nucleic acids,
including degenerate nucleic acids, encoding the disclosed variants
and derivatives of the polypeptide sequences. Thus, while each
particular nucleic acid sequence may not be written out herein, it
is understood that each and every sequence is in fact disclosed and
described herein through the disclosed polypeptide sequence.
Provided herein are compounds to be tested for their ability to
bind and/or inhibit SENP1. As used herein, an inhibitor refers to
an agent or compound that inhibits SENP1 directly or indirectly.
For example, an inhibitor of SENP1 can inhibit the expression or
activity of SENP1. Compounds to be tested in the provided methods
include, but are not limited to, small molecules, peptides, nucleic
acids and antibodies. Optionally, the compound to be tested is a
small molecule. Optionally, the small molecule is an inhibitor of
SENP1. Small molecule inhibitors of SENP1 include, but are not
limited to the compounds in Tables 1 and 2 and those described in
WO 2012/064887, which is incorporated by reference herein in its
entirety. As used herein, the term "small molecule" refers to an
organic compound containing carbon. A small molecule is generally,
but not necessarily, of low molecular weight, e.g., less than 1000
Daltons.
Once a compound has been identified as binding to SENP1 and/or
inhibiting SENP1, the compound can be further tested for its
binding and/or inhibitory abilities using a variety of known
methods including the methods described in the example below.
Various assays for determining levels and activities of protein are
available, such as amplification/expression methods,
immunohistochemistry methods, FISH and shed antigen assays,
southern blotting, or PCR techniques. Moreover, the protein
expression or amplification may be evaluated using in vivo
diagnostic assays.
Compositions and Apparatuses for NMR Analysis
Provided herein are compositions comprising a SENP1 polypeptide and
NMR apparatuses comprising the compositions for NMR analysis.
Optionally, the composition is an aqueous solution. Optionally, the
aqueous solution comprises an SENP1 polypeptide at a pH from about
6.0 to about 7.5. For example, the pH can be about 6.8. The
provided compositions or aqueous solutions can further include, for
example, buffering agents, reducing agents, solvents, bases and
combinations thereof. Buffering agents include, but are not limited
to, phosphate or citrate buffers. Reducing agents include but are
not limited to, dithiothreitol, and sodium borohydride. Bases
include, but are not limited to, metal oxides and salts of
carbanions, amides and hydrides. Solvents include, but are not
limited to, dimethyl sulfoxide (DMSO) Optionally, the compositions
can include sodium phosphate, DMSO, D.sub.2O, sodium azide,
dithiothreitol or combinations thereof. By way of example, the
sodium phosphate can be present at about 20 mM. Optionally, the
SENP1 polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO complex. Optionally, the SENP1 polypeptide is bound to a
compound thereby forming a SENP1-compound complex. Optionally, the
SENP1 polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO-compound complex. Optionally, the SUMO protein is a
truncated SUMO protein. Optionally, the SENP1 polypeptide comprises
SEQ ID NO:1, 2, 3, 4, 5, 6, or 7. Optionally, the SENP1 polypeptide
comprises amino acid residues 440-455, 463-473, 493-515, 529-535,
550-554, or 596-603 numbered relative to SEQ ID NO:1.
An NMR apparatus comprising an NMR sample container for NMR
analysis, said NMR sample container comprising the aqueous
composition or solution is also provided. NMR apparatuses are known
and can be obtained from commercially available sources. Makers of
NMR equipment include, but are not limited to, Bruker (Germany),
Oxford Instruments (United Kingdom), General Electric (Fairfield,
Conn.), Philips (Amsterdam, Netherlands), Siemens AG (Munich,
Germany) and Agilent Technologies, Inc. (Santa Clara, Calif.).
Compositions
Provided herein are compositions including the inhibitors
identified by the screening and binding methods provided herein.
The compositions are, optionally, suitable for formulation and
administration in vitro or in vivo. Optionally, the compositions
comprise one or more of the provided agents and a pharmaceutically
acceptable carrier. Suitable carriers and their formulations are
described in Remington: The Science and Practice of Pharmacy,
21.sup.st Edition, David B. Troy, ed., Lippicott Williams &
Wilkins (2005). By pharmaceutically acceptable carrier is meant a
material that is not biologically or otherwise undesirable, i.e.,
the material is administered to a subject without causing
undesirable biological effects or interacting in a deleterious
manner with the other components of the pharmaceutical composition
in which it is contained. If administered to a subject, the carrier
is optionally selected to minimize degradation of the active
ingredient and to minimize adverse side effects in the subject.
The inhibitors are administered in accord with known methods, such
as intravenous administration, e.g., as a bolus or by continuous
infusion over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, intratumoral or inhalation routes. The
administration may be local or systemic. The compositions can be
administered via any of several routes of administration, including
topically, orally, parenterally, intravenously, intra-articularly,
intraperitoneally, intramuscularly, subcutaneously, intracavity,
transdermally, intrahepatically, intracranially,
nebulization/inhalation, or by installation via bronchoscopy. Thus,
the compositions are administered in a number of ways depending on
whether local or systemic treatment is desired, and on the area to
be treated.
The compositions for administration will commonly comprise an agent
as described herein (e.g. inhibitor of SENP1) dissolved in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers can be used, e.g., buffered saline
and the like. These solutions are sterile and generally free of
undesirable matter. These compositions may be sterilized by
conventional, well known sterilization techniques. The compositions
may contain pharmaceutically acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of active agent in these formulations can vary
widely, and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the subject's
needs.
Pharmaceutical formulations, particularly, of the modified viruses
can be prepared by mixing the modified adenovirus (or one or more
nucleic acids encoding the modified adenovirus) having the desired
degree of purity with optional pharmaceutically acceptable
carriers, excipients or stabilizers. Such formulations can be
lyophilized formulations or aqueous solutions.
Acceptable carriers, excipients, or stabilizers are nontoxic to
recipients at the dosages and concentrations used. Acceptable
carriers, excipients or stabilizers can be acetate, phosphate,
citrate, and other organic acids; antioxidants (e.g., ascorbic
acid) preservatives low molecular weight polypeptides; proteins,
such as serum albumin or gelatin, or hydrophilic polymers such as
polyvinylpyllolidone; and amino acids, monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents; and ionic and non-ionic surfactants
(e.g., polysorbate); salt-forming counter-ions such as sodium;
metal complexes (e.g. Zn-protein complexes); and/or non-ionic
surfactants. The modified adenovirus (or one or more nucleic acids
encoding the modified adenovirus) can be formulated at any
appropriate concentration of infectious units.
Formulations suitable for oral administration can consist of (a)
liquid solutions, such as an effective amount of the modified
adenovirus suspended in diluents, such as water, saline or PEG 400;
(b) capsules, sachets or tablets, each containing a predetermined
amount of the active ingredient, as liquids, solids, granules or
gelatin; (c) suspensions in an appropriate liquid; and (d) suitable
emulsions. Tablet forms can include one or more of lactose,
sucrose, mannitol, sorbitol, calcium phosphates, corn starch,
potato starch, microcrystalline cellulose, gelatin, colloidal
silicon dioxide, talc, magnesium stearate, stearic acid, and other
excipients, colorants, fillers, binders, diluents, buffering
agents, moistening agents, preservatives, flavoring agents, dyes,
disintegrating agents, and pharmaceutically compatible carriers.
Lozenge forms can comprise the active ingredient in a flavor, e.g.,
sucrose, as well as pastilles comprising the active ingredient in
an inert base, such as gelatin and glycerin or sucrose and acacia
emulsions, gels, and the like containing, in addition to the active
ingredient, carriers known in the art.
The inhibitors of SENP1 can be made into aerosol formulations
(i.e., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like.
Formulations suitable for parenteral administration, such as, for
example, by intraarticular (in the joints), intravenous,
intramuscular, intratumoral, intradermal, intraperitoneal, and
subcutaneous routes, include aqueous and non-aqueous, isotonic
sterile injection solutions, which can contain antioxidants,
buffers, bacteriostats, and solutes that render the formulation
isotonic with the blood of the intended recipient, and aqueous and
non-aqueous sterile suspensions that can include suspending agents,
solubilizers, thickening agents, stabilizers, and preservatives. In
the provided methods, compositions can be administered, for
example, by intravenous infusion, orally, topically,
intraperitoneally, intravesically intratumorally, or intrathecally.
Parenteral administration, intratumoral administration, and
intravenous administration are the preferred methods of
administration. The formulations of compounds can be presented in
unit-dose or multi-dose sealed containers, such as ampules and
vials.
Injection solutions and suspensions can be prepared from sterile
powders, granules, and tablets of the kind previously described.
Cells transduced or infected by adenovirus or transfected with
nucleic acids for ex vivo therapy can also be administered
intravenously or parenterally as described above.
The pharmaceutical preparation is preferably in unit dosage form.
In such form the preparation is subdivided into unit doses
containing appropriate quantities of the active component. Thus,
the pharmaceutical compositions can be administered in a variety of
unit dosage forms depending upon the method of administration. For
example, unit dosage forms suitable for oral administration
include, but are not limited to, powder, tablets, pills, capsules
and lozenges.
Methods of Treatment
The provided inhibitors of SENP1 can be administered for
therapeutic or prophylactic treatments or used in the laboratory.
Thus, provided is a method of treating a proliferative disorder in
a subject. The method includes administering the provided
inhibitors of SENP1 or compositions to the subject. As described
throughout, the pharmaceutical composition is administered in any
number of ways including, but not limited to, intravenously,
intravascularly, intrathecally, intramuscularly, subcutaneously,
intraperitoneally, or orally. Optionally, the method further
comprising administering to the subject one or more additional
therapeutic agents. Optionally, the therapeutic agent is a
chemotherapeutic agent.
As described throughout, the proliferative disorder can be cancer.
Optionally, the proliferative disorder is selected from the group
consisting of lung cancer, prostate cancer, colorectal cancer,
breast cancer, thyroid cancer, renal cancer, liver cancer and
leukemia. Optionally, the proliferative disorder is metastatic.
In therapeutic applications, compositions are administered to a
subject suffering from a proliferative disease or disorder (e.g.,
cancer) in a "therapeutically effective dose." Amounts effective
for this use will depend upon the severity of the disease and the
general state of the patient's health. Single or multiple
administrations of the compositions may be administered depending
on the dosage and frequency as required and tolerated by the
patient. A "patient" or "subject" includes both humans and other
animals, particularly mammals. Thus the methods are applicable to
both human therapy and veterinary applications.
Optionally, the provided methods include administering to the
subject one or more additional therapeutic agents. Thus, the
provided methods can be combined with other cancer therapies,
radiation therapy, hormone therapy, or chemotherapy. The combined
administrations contemplates coadministration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Combinations of
agents or compositions can be administered either concomitantly
(e.g., as a mixture), separately but simultaneously (e.g., via
separate intravenous lines) or sequentially (e.g., one agent is
administered first followed by administration of the second agent).
Thus, the term combination is used to refer to concomitant,
simultaneous or sequential administration of two or more agents or
compositions.
According to the methods provided herein, the subject is
administered an effective amount of one or more of the agents
provided herein. The terms effective amount and effective dosage
are used interchangeably. The term effective amount is defined as
any amount necessary to produce a desired physiologic response
(e.g., killing of a cancer cell). The dosages, however, may be
varied depending upon the requirements of the subject, the severity
of the condition being treated, and the compound being employed.
For example, dosages can be empirically determined considering the
type and stage of cancer diagnosed in a particular subject. The
dose administered to a subject, in the context of the provided
methods should be sufficient to affect a beneficial therapeutic
response in the patient over time. Determination of the proper
dosage for a particular situation is within the skill of the
practitioner. Thus, effective amounts and schedules for
administering the agent may be determined empirically by one
skilled in the art. The dosage ranges for administration are those
large enough to produce the desired effect in which one or more
symptoms of the disease or disorder are affected (e.g., reduced or
delayed). The dosage should not be so large as to cause substantial
adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will
vary with the age, condition, sex, type of disease, the extent of
the disease or disorder, route of administration, or whether other
drugs are included in the regimen, and can be determined by one of
skill in the art. The dosage can be adjusted by the individual
physician in the event of any contraindications. Dosages can vary
and can be administered in one or more dose administrations daily,
for one or several days. Guidance can be found in the literature
for appropriate dosages for given classes of pharmaceutical
products.
As used herein the terms treatment, treat, or treating refers to a
method of reducing the effects of one or more symptoms of a disease
or condition characterized by expression of the protease or symptom
of the disease or condition characterized by expression of the
protease. Thus in the disclosed method, treatment can refer to a
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in
the severity of an established disease, condition, or symptom of
the disease or condition. For example, a method for treating a
disease is considered to be a treatment if there is a 10% reduction
in one or more symptoms of the disease in a subject as compared to
a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10%
and 100% as compared to native or control levels. It is understood
that treatment does not necessarily refer to a cure or complete
ablation of the disease, condition, or symptoms of the disease or
condition. Further, as used herein, references to decreasing,
reducing, or inhibiting include a change of 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90% or greater as compared to a control level
and such terms can include but do not necessarily include complete
elimination.
Kits
Provided herein are kits for screening for compounds that bind or
inhibit SENP1. The kits include a composition comprising an SENP1
polypeptide. Optionally, the composition is an aqueous solution.
Optionally, the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4,
5, 6, or 7. Optionally, the SENP1 polypeptide comprises amino acid
residues 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603
numbered relative to SEQ ID NO:1. Optionally, the aqueous
composition comprising an SENP1 polypeptide is at a pH from about
6.0 to about 7.5. Optionally, the pH is about 6.8. Optionally, the
compositions can further include, for example, buffering agents,
reducing agents, bases and combinations thereof. Optionally, the
compositions can include sodium phosphate, D.sub.2O, sodium azide,
dithiothreitol or combinations thereof. By way of example, the
sodium phosphate can be present at about 20 mM. Optionally, the
SENP1 polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO complex. Optionally, the SENP1 polypeptide or SENP1-SUMO
complex is bound to a compound thereby forming a SENP1-compound
complex or SENP1-SUMO-compound complex. Optionally, the SUMO
protein is a truncated SUMO protein. In some embodiments, the kit
comprises a container including a SENP1 polypeptide or SENP1-SUMO
complex and, optionally, a second container including a
SENP1-compound complex or SENP-SUMO-compound complex.
Further provided are kits including an inhibitor of SENP1.
Optionally, the kit comprises one or more doses of an effective
amount of a composition comprising a SENP1 inhibitor. Optionally,
the composition is present in a container (e.g., vial or packet).
Optionally, the kit further includes one or more additional
therapeutic agents. Optionally, the therapeutic agent is a
chemotherapeutic agent. Optionally, the kit comprises a means of
administering the composition, such as, for example, a syringe,
needle, tubing, catheter, patch, and the like. The kit may also
comprise formulations and/or materials requiring sterilization
and/or dilution prior to use.
Disclosed are materials, compositions, and components that can be
used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed methods and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly
disclosed, each is specifically contemplated and described herein.
For example, if a method is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the method are discussed, each and every combination and
permutation of the method, and the modifications that are possible
are specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. This concept applies to
all aspects of this disclosure including, but not limited to, steps
in methods using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed, it is understood
that each of these additional steps can be performed with any
specific method steps or combination of method steps of the
disclosed methods, and that each such combination or subset of
combinations is specifically contemplated and should be considered
disclosed.
Publications cited herein and the material for which they are cited
are hereby specifically incorporated by reference in their
entireties.
A number of embodiments have been described. Nevertheless, it will
be understood that various modifications may be made. Accordingly,
other embodiments are within the scope of the claims.
EXAMPLE
Example 1. Identification and Characterization of a SENP
Inhibitors
Enzymes called SENPs catalyze both the maturation of small
ubiquitin-like modifier (SUMO) precursors and removal of SUMO
modifications, which regulate essential cellular functions such as
cell cycle progression, DNA damage response and intracellular
trafficking. Some members, such as SENP1, are potential targets for
developing cancer therapeutics. A search for small molecule
inhibitors of SENPs was carried out using in-silico screening in
conjunction with biochemical assays, and a new chemotype of small
molecule inhibitors that non-covalently inhibit SENPs was
identified. The inhibitors confer the non-competitive inhibitory
mechanism, as shown by nuclear magnetic resonance (NMR) and
quantitative enzyme kinetic analysis. The NMR data also provided
evidence for substrate-assisted inhibitor binding, which indicates
the need for caution in using artificial substrates for compound
screening, as the inhibitory effects could be significantly
different from using the physiological substrates.
In this study, it was purported to identify small molecule
inhibitors of SENPs through in-silico screening in conjunction with
enzyme kinetic, nuclear magnetic resonance (NMR) and cellular
analyses. In silico screening was performed using Protein Data Bank
(PDB) accession codes 2IYC and 2IY1 and by considering hydrogen
bonding and hydrophobic interactions between the C-terminus of
full-length SUMO-1 and SENP1. The GLIDE program (Friesner et al.,
Journal of Medicinal Chemistry 47:1739-1749 (2004)) was used to
search the 250,000 compound library provided by the Developmental
Therapeutics Program (DTP) of the National Cancer Institute, using
the E-model scoring function of Cvdw, which is the sum of the van
der Waals (Evdw) and electrostatic interaction energy terms
(Eelec). Among the top hits, the dominant scaffolds were
peptidomimetics and compounds that contained 2-fold symmetry. Forty
compounds (100 .mu.M) representing the dominant scaffolds were
tested for their inhibitory effects on SENP1 and SENP2 for
maturation of SUMO-1 and SUMO-2 precursors. The most potent
compounds contained sulfonyl-benzene groups. Additional analogues
of this group were obtained from DTP, and NSC5068, hereafter
referred to as SPI-01 (SUMO protease inhibitor), was found to have
the highest potency (Table 1). Available analogs of SPI-01 were
obtained from DTP. Five compounds in this group (Table 1, SPI-06 to
SPI-10) are "half" of the other compounds (Table 1, SPI-01 to
SPI-05) and allowed the exploration of the activity requirements of
the two-fold symmetric structure of SPI-01 to SPI-05. The
inhibitory activity of these compounds on SENP1 and SENP2 was
characterized using substrates that contained precursor SUMO-1 or
SUMO-2 (S) flanked by yellow fluorescent protein (Y) at the
N-terminus and enhanced cyan fluorescent protein (E) at the
C-terminus (YSE) (Tatham and Hay, Methods Mol. Biol. 497:253-268
(2009)). Although the cleavage of the substrates can be detected by
fluorescence resonance energy transfer (FRET), FRET could not be
used because many of these compounds interfere with the FRET
signal. Therefore, a gel-based assay was used to determine the
inhibitory effects of all compounds on SENP1 and 2 (representative
data shown in FIGS. 1 and 2), and the gel bands were quantified to
determine the half maximum inhibitory concentrations (IC.sub.50)
(Table 1). The inhibitory effects of the compounds on the
endopeptidase activities were not only enzyme-dependent, but also
substrate-dependent. For SENP1-mediated cleavage of SUMO-1
precursor, only four of the compounds (SPI-01 to SPI-04) had half
maximal inhibitory concentrations (IC.sub.50) below 60 .mu.M. The
inhibitors were more potent for inhibiting SENP2 than SENP1 for
cleavage of the SUMO-1 precursor. However, for cleavage of the
SUMO-2 precursor, some compounds (i.e. SPI-01 and SPI-04) had
similar potency for inhibiting SENP1 and SENP2, while others (i.e.
SPI-07 and SPI-10) were more potent for inhibiting SENP1 than SENP2
or vice versa (i.e. SPI-06 and SPI-09) (Table 1). In addition to
the differential effects on SENP1 and SENP2, SPI-01 had more than
10 fold less potency for inhibiting a de-ubiquitin enzyme
isopeptidase T than inhibiting SENP2.
TABLE-US-00001 TABLE 1 Compounds IC.sub.50 (.mu.M)-SUMO1 IC.sub.50
(.mu.M)-SUMO2 Structure Code.dagger. NCI ID.dagger-dbl. SENP1 SENP2
SENP1 SENP2 ##STR00001## SPI-01 NSC5068 32.8 .+-. 1.82 1.42 .+-.
3.0 1.88 .+-. 2.2 1.1 .+-. 5.8 ##STR00002## SPI-02 NSC16224 26.5
.+-. 1.86 3.42 .+-. 1.6 2.08 .+-. 2.0 2.70 .+-. 2.1 ##STR00003##
SPI-03 NSC8676 20.27 .+-. 2.47 5.17 .+-. 1.32 1.86 .+-. 2.3 3.0
.+-. 2.0 ##STR00004## SPI-04 NSC34933 11.2 .+-. 1.7 1.6 .+-. 2.5
2.32 .+-. 2.6 2.15 .+-. 2.28 ##STR00005## SPI-05 NSC5067 >60
19.7 .+-. 1.47 7.5 .+-. 1.6 4.6 .+-. 1.65 ##STR00006## SPI-06
NSC70551 >60 3.62 .+-. 1.98 43.2 .+-. 2.2 10.7 .+-. 1.6
##STR00007## SPI-07 NSC58046 >60 >60 17.54 .+-. 4.9 28.06
.+-. 9.2 ##STR00008## SPI-08 NSC22940 >60 4.1 .+-. 3.0 >60
41.06 .+-. 5.2 ##STR00009## SPI-09 NSC42164 >60 23.36 .+-. 1.6
>60 26.96 .+-. 2.5 ##STR00010## SPI-10 NSC45551 >60 34.21
.+-. 1.9 11.1 .+-. 3.7 36.44 .+-. 5.7
To determine whether other SENPs can be inhibited by this family of
inhibitors, a distant SENP member, SENP7, was tested in parallel
with SENP1 and SENP2 using a pentapeptide substrate that contained
the Gly-Gly motif and luciferin, known as DUB-Glo (Promega,
Madison, Wis.). Cleavage of luciferin by a SENP can be detected by
a coupled bioluminescent assay using luciferase. The bioluminescent
reporter was chosen instead of a fluorescent reporter to avoid
interference by the compounds during detection. In addition,
because SENP7 has different physiological substrates than SENP1 and
SENP2 (Kolli et al., Biochemical Journal 430:335-344 (2010); and
Shen et al., EMBO Rep. 13(4):339-46 (2012)), an advantage of
DUB-Glo is that it can act as a common substrate for all SENPs,
which enabled us to rule out substrate-specific effects. The
dose-dependent inhibition of each SENP by the inhibitors was
determined (FIG. 3), as was the IC.sub.50 for inhibition of SENP1,
2 and 7 of all the compounds (Table 2). Most compounds had more
similar inhibitory effects on SENP1 and SENP2 than on SENP7,
consistent with their amino acid sequence similarities. In
addition, the compounds were more potent for inhibiting SENP1 when
DUB-Glo was used as a substrate than when SUMO-1 precursor was used
(Tables 1 and 2). To rule out the possibility that these compounds
used a promiscuous mechanism, the compounds were also tested in
SUMOylation and ubiquitination reactions, which also depend on
enzymes containing catalytic Cys residues. The compounds were
noninhibitory in these assays. Furthermore, comparison of the
DUB-Glo and the SUMO maturation assays revealed that the effect of
SENP inhibitors could be highly substrate-specific.
TABLE-US-00002 TABLE 2 SENP1 SENP2 SENP7 Structure Code.dagger. NCI
ID.dagger-dbl. IC.sub.50 (.mu.M) IC.sub.50 (.mu.M) IC.sub.50
(.mu.M) ##STR00011## SPI-01 NSC5068 5.9 .+-. 1.4 2.9 .+-. 1.6 3.5
.+-. 1.5 ##STR00012## SPI-02 NSC 16224 2.1 .+-. 1.9 2.0 .+-. 2.0
2.7 .+-. 1.8 ##STR00013## SPI-03 NSC 8676 3.8 .+-. 1.5 2.4 .+-. 1.8
4.8 .+-. 1.4 ##STR00014## SPI-04 NSC 34933 2.4 .+-. 1.8 2.3 .+-.
1.8 3.4 .+-. 1.5 ##STR00015## SPI-05 NSC 5067 13.3 .+-. 1.3 8.5
.+-. 1.3 4.6 .+-. 1.5 ##STR00016## SPI-06 NSC 70551 3.9 .+-. 1.4
3.7 .+-. 1.4 4.7 .+-. 1.7 ##STR00017## SPI-07 NSC 58046 >>60
>>60 1.9 .+-. 2.2 ##STR00018## SPI-08 NSC 22940 22.2 .+-. 1.5
17.2 .+-. 1.5 2.8 .+-. 1.6 ##STR00019## SPI-09 NSC 42164 >60 6.8
.+-. 1.3 1.9 .+-. 2.1 ##STR00020## SPI-10 NSC 45551 2.4 .+-. 1.8
2.5 .+-. 1.7 2.0 .+-. 2.0
The abilities of representative inhibitors were then tested to
inhibit SENP in cells. HeLa cells were treated with increasing
concentrations of SPI-01 for 48 hours, after which SUMOylated
proteins were detected in the cells by Western blots. SUMO-2/3
conjugates accumulated in cells and this accumulation correlated
with inhibitor concentration, particularly at high molecular
weights (FIG. 4). This result suggests that SPI-01 inhibits the
isopeptidase activities of SENPs, particularly SENP6 and SENP7,
which are required for SUMO chain editing. It was observed that
less significant effects on the accumulation of SUMO-1 conjugates,
possibly because most SENPs cleave SUMO-2/3-conjugates. It is known
that heat shock triggers a dramatic increase in global SUMO-2/3
conjugations and that during recovery, the SUMOylated proteins are
removed, at least in part, due to the deSUMOylation activity of
SENP1 (Nefkens et al., J. Cell Sci. 116:513-524 (2003)). To further
confirm that the inhibitors inhibited deSUMOylase activities, HeLa
cells were treated with SPI-01 and SPI-02 for 2 hours at 37.degree.
C. Then, SPI-treated or untreated control HeLa cells was
transferred to 42.degree. C. for 30 minutes, followed by recovery
for 4 hours at 37.degree. C. before processing for detection of
global SUMO-2/3 levels. The inhibitor-treated cells had
considerably higher levels of SUMOylated proteins than did the
corresponding controls that did not receive heat shock or the
mock-treated cells after the recovery period (FIG. 5). Thus, the
results of the heat-shock experiments further confirmed that the
SPI compounds had inhibitory effects on SENPs in cells.
NMR chemical shift perturbation (CSP) analysis was used to
investigate whether this family of inhibitors binds the enzyme or
the enzyme-substrate complex. CSP experiments were conducted using
a .sup.15N-labeled C603S mutant of the human SENP1 catalytic domain
(SENP1-C603S, for which NMR chemical shift assignments have been
obtained and deposited in the Biological Magnetic Resonance Bank
(BMRB) with accession number 19083). Although the SENP1-C603S
mutant is catalytically inactive (Xu et al., Biochem. J. 398:345-52
(2006)), it retains binding activity for the precursor or mature
SUMO paralogs or SUMOylated substrates (Shen et al., Nat. Struct.
Mol. Biol. 13:1069-1077 (2006)). It was observed that SPI-01 caused
modest backbone amide CSP for a subset of SENP1-C603S residues. Of
note, specific CSPs were observed at the canonical
cysteine-protease catalytic triad residues (D550, H533, and C603),
the proposed dynamic channel of conserved W465 and W534, and at
several other residues located at or adjacent to the SENP catalytic
center (W465, L466, G531, H533, W534, C535, M552, G554 and Q596)
with only one residue located distal to this surface (E469) (FIG.
2A). Interestingly, M552, G554, and Q596 are clustered at the SENP1
surface that contacts the C-terminal tail of SUMO-1. Supporting the
importance of this surface in SENP catalytic activity,
non-conservative point mutations of Q596 in SENP1 or the equivalent
residue to SENP1 M552 in SENP2 (M497) perturb SUMO processing and
deconjugation (Reverter and Lima, Nat. Struct. Mol. Biol. 13;
1060-8 (2006); and Shen et al., Biochem. J. 397:279-288 (2006)).
Residue E469 is positioned toward the binding surface for the
structured region of SUMO-1, and its CSP may be due to an
alternative interaction with the compound or long-range effects.
These results indicate that SPI-01 binds the surface adjacent to
the catalytic center that contacts the C-terminal portion of the
SUMO precursors. The residues that showed CSP are highly conserved
between SENP1 and SENP2, suggesting that SPI-01 can interact with
the equivalent surface on SENP2.
The binding of SPI-01 to the enzyme-substrate complex was
investigated. CSP analysis was carried out on the 40 kDa complex of
.sup.15N-labeled full length precursor SUMO-1-GGHSTV (SUMO-1-FL)
with unlabeled SENP1-C603S. An equimolar amount of SPI-01 was added
to the 1:1 enzyme-substrate complex. The only observed CSP on the
.sup.15N-labeled precursor SUMO-1-FL was on the C-terminal residues
S99 and V101 (FIGS. 7 and 8) (Song et al., PNAS 101:14373-8
(2004)). This result indicates that SPI-01 binds the
enzyme-substrate complex at the interface between SENP and the
C-terminal tails of precursor SUMO-FL. X-ray crystal structures
showed that the C-terminal tail of precursor SUMO sits in and
projects out of the catalytic tunnel of SENPs (Shen et al., Nat.
Struct. Mol. Biol. 13:1069-77 (2006)). In the case of SENP1, the
region that interacts with the projected C-terminus is
predominantly acidic and favors the C-terminus of SUMO-1, which is
polar and positively charged, over that of SUMO-2, whose C-terminus
is mainly hydrophobic (Shen et al., Nat. Struct. Mol. Biol.
13:1069-77 (2006); and Shen et al., The Biochemical Journal
397:279-88 (2006)). In addition, the more hydrophobic C-terminus of
SUMO-2 may favor binding of aromatic inhibitors. These properties
may account for the more potent inhibition of processing of the
SUMO-2 precursor (Table 1).
To further investigate the inhibitory mechanism, enzyme kinetic
experiments were conducted using the pentapeptide substrate DUB-Glo
(FIG. 9). The data was fit to a mixed inhibition mechanism, as
described by the kinetic equation:
.times..times..function..alpha..times..times..times..function..function..-
alpha..times..times. ##EQU00001##
in which the value of ".alpha." indicates the mechanism of
inhibition (Segel, Enzyme Kinetics John Wiley & Sons (1993)).
For both SENP1 and SENP2, the ".alpha." values indicated that the
inhibitory mechanism is mainly noncompetitive and suggests that the
inhibitor binds to the enzyme and the enzyme-substrate complex to
inhibit chemical conversion. This finding is consistent with the
NMR binding analysis indicating that the inhibitor binds both the
enzyme and the enzyme-substrate complex as discussed above.
In conclusion, this study has identified SENP inhibitors that do
not covalently modify the catalytic Cys residue. This study has
also provided the first mechanistic insights into how a small
molecule inhibitor of SENPs that does not covalently modify the
catalytic Cys can inhibit the enzymes. The substrate-assisted
inhibitor binding indicates the need for caution in designing high
throughput screening assays that use fluorogenic or
chemiluminescent artificial substrates, as the results could be
significantly different from using the physiological substrates.
The substrate-dependent inhibitory effect suggests the possibility
of designing SENP inhibitors that are tuned for
substrate-specificity.
Materials and Methods
Protein Purification. The catalytic domains of SENP1, 2, and 7 were
expressed as His-tagged protein in E. coli (DE3) and purified using
nickel affinity chromatography (Namanja et al., The Journal of
Biological Chemistry 287:3231-3240 (2012)). The pET11 expression
plasmids for SENP1 and 2 contained a cDNA insert coding for the
catalytic domain of human SENP1-WT (419-644) and SENP2-WT
(364-589). The expression plasmid for the SENP1 active site point
mutant C603S was generated using the QuikChange mutagenesis kit
(Agilent Technologies, San Diego, Calif.). The expression plasmid
for the catalytic domain of SENP7 has been described (Mikolajczyk
et al., Journal of Biological Chemistry 282:26217-26224
(2007)).
SUMO Cleavage Assays. SUMO cleavage assays were performed by
incubating SENPs with various concentrations of the inhibitor (0-60
.mu.M) at room temperature for 10 min in assay buffer (50 mM Tris,
pH 7.4, 100 mM NaCl, 10 mM DTT). SENP concentrations were 32-50 nM
when 50 .mu.g/ml of the final substrate YFP-SUMO-ECFP (YSE) fusion
protein was added. The mixture was incubated (37.degree. C., 15
min), followed by SDS-PAGE and Coomassie staining for
visualization. For cellular SENP inhibition experiments, HeLa cells
cultured in DMEM plus 10% FBS, 100 units/ml penicillin, 100 mg/ml
streptomycin, and 0.2 M glutamine were treated for 48 hours with
SPI compounds. For heat shock experiment, HeLa cells were treated
with SPI compounds or mock treated (2 h, 37.degree. C.), after
which cells were transferred to 42.degree. C. for 30 min. After
heat shock, the cells were allowed to recover (4-5 hours) before
being harvested and lysed. Proteins were separated by SDS-PAGE and
immunoblotted to determine global SUMO-2/3 levels.
DUB-Glo Assay. The luciferase substrate assay (DUB-Glo, Promega,
Madison, Wis.) was performed according to the manufacturer's
instructions. Briefly, SENPs (final concentration 50-100 nM) in
Tris buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 10 mM DTT) were
pre-incubated (10 min, room temperature) with increasing
concentrations of inhibitor (0-60 .mu.M final concentration)
followed by addition of the luciferase substrate. Luciferase output
was recorded 30 min after addition of the luciferase substrate.
Values are the averages of experiments performed in triplicate.
NMR Experiments. Samples used for NMR titration or chemical shift
perturbation analyses were .sup.15N or .sup.15N/.sup.13C-labeled;
the titrant protein or SPI-01 was not labeled. The
.sup.15N/.sup.13C SUMO-1-FL sample was used to extend the backbone
assignments of mature SUMO-1 to the HSTV tail by using
2D-.sup.15N-.sup.1H-HSQC, 3D-HNCA, 3D-HNCOCA, and 3D-HNCACB.
Additionally, comparison of .sup.15N-.sup.1H-HSQC between precursor
and mature SUMO quickly identified the resonances of the HSTV tail.
For SENP1 assignments, a full suite of triple-resonance NMR
experiments were acquired on .sup.15N/.sup.13C/.sup.2H or
.sup.15N/.sup.13C samples: HNCA, HNCOCA, HNCACB, HNCOCACB, HNCO,
HNCACO, and NOESY-HSQC. All samples were dissolved in the NMR
buffer: 20 mM sodium phosphate (pH 6.8), 10% D2O, 0.03% sodium
azide and 10 mM d10-dithiothreitol. Purified perdeuterated SENP1
samples were unfolded and refolded into NMR buffer.
For titration of SENP1-C603S with SPI-01, 270 .mu.M
.sup.15N-labeled sample was titrated with the inhibitor that was
prepared by diluting a 10 mM stock in 100% DMSO-d.sub.6 to a
concentration of 1.7 mM in the NMR buffer. The 2D
.sup.1H-.sup.15N-HSQC spectra of SENP1 were recorded at each
incremental addition of 5 .mu.l of SPI-01 into 250 .mu.l of SENP1.
The chemical shift perturbation (CSP) analysis compared the spectra
of SENP1 in the absence or the presence of equimolar SPI-01. A
separate DMSO control titration was performed to account for
DMSO-induced CSP. NMR resonance assignments for SUMO samples at
35.degree. C. were transferred from those obtained at 25.degree. C.
by spectral acquisition at 2.5.degree. C. incremental increases.
All data were acquired on a 600 MHz Bruker Avance NMR spectrometer
equipped with a TXI Cryoprobe.
TABLE-US-00003 TABLE 3 Free SENP1 NMR Chemical Shifts Values.
Chemical Shift Ambiguity Index Value Definitions The values other
than 1 are used for those atoms with different chemical shifts that
cannot be assigned to stereospecific atoms or to specific residues
or chains. Index Value Definition 1 Unique (including isolated
methyl protons germinal atoms, and geminal methyl groups with
identical chemical shifts (e.g. ILE HD11, HD12, HD13 protons) 2
Ambiguity of geminal atoms or geminal methyl proton groups (e.g.
ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, or LEU HD11,
HD12, HD13 and HD21, HD22, HD23 methyl protons) 3 Aromatic atoms on
opposite sides of symmetrical rings (e.g. TYR HE1 and HE2 protons)
4 Intraresidue ambiguities (e.g. LYS HG and HD protons or TRP HZ2
and HZ3 protons) 5 Interresidue ambiguities (LYS 12 vs. LYS 27) 6
Intermolecular ambiguities (e.g. ASP 31 CA in monomer 1 and ASP 31
CA in monomer 2 of an asymmetrical homodimer, duplex DNA
assignments, or other assignments that may apply to atoms in one or
more molecule in the molecular assembly) 9 Ambiguous, specific
ambiguity not defined Chemical Atom Residue Amino Atom Atom Iso-
shift Unique- number number acid context type type (ppm)* ness 1
419 E CA C 13 56.635 1 2 419 E CB C 13 29.326 1 3 419 E CO C 13
175.803 1 4 419 E H H 1 8.056 1 5 419 E N N 15 120.257 1 6 420 F CA
C 13 54.951 1 7 420 F CB C 13 37.882 1 8 420 F CO C 13 173.207 1 9
420 F H H 1 8.035 1 10 420 F N N 15 118.648 1 11 422 E CA C 13
56.498 1 12 422 E CB C 13 29.476 1 13 422 E CO C 13 176.111 1 14
422 E H H 1 8.637 1 15 422 E N N 15 124.065 1 16 423 I CA C 13
60.725 1 17 423 I CB C 13 35.427 1 18 423 I CO C 13 176.659 1 19
423 I H H 1 8.522 1 20 423 I N N 15 122.041 1 21 424 T CB C 13
70.292 1 22 424 T CO C 13 174.725 1 23 424 T H H 1 7.633 1 24 424 T
N N 15 121.188 1 25 425 E CA C 13 59.696 1 26 425 E CB C 13 28.462
1 27 425 E H H 1 8.913 1 28 425 E N N 15 120.9 1 29 426 E CA C 13
59.444 1 30 426 E CO C 13 179.787 1 31 426 E H H 1 8.419 1 32 426 E
N N 15 118.024 1 33 427 M CB C 13 33.371 1 34 427 M CO C 13 177.912
1 35 427 M H H 1 7.366 1 36 427 M N N 15 119.301 1 37 428 E CB C 13
28.41 1 38 428 E CO C 13 178.828 1 39 428 E H H 1 8.605 1 40 428 E
N N 15 118.58 1 41 429 K CA C 13 59.488 1 42 429 K CB C 13 31.407 1
43 429 K CO C 13 178.978 1 44 429 K H H 1 7.858 1 45 429 K N N 15
118.101 1 46 430 E CB C 13 29.584 1 47 430 E CO C 13 179.111 1 48
430 E H H 1 7.356 1 49 430 E N N 15 119.087 1 50 431 I CA C 13
64.567 1 51 431 I CB C 13 38.123 1 52 431 I CO C 13 176.796 1 53
431 I H H 1 8.075 1 54 431 I N N 15 119.774 1 55 432 K CA C 13
59.195 1 56 432 K CB C 13 31.197 1 57 432 K CO C 13 180.185 1 58
432 K H H 1 8.32 1 59 432 K N N 15 116.686 1 60 433 N CA C 13
55.822 1 61 433 N CB C 13 37.95 1 62 433 N CO C 13 178.466 1 63 433
N H H 1 7.635 1 64 433 N N N 15 114.913 1 65 434 V CA C 13 64.189 1
66 434 V CB C 13 30.396 1 67 434 V CG1 C 13 22.475 1 68 434 V CG2 C
13 21.674 1 69 434 V CO C 13 176.412 1 70 434 V H H 1 7.548 1 71
434 V HG1 H 1 0.724 1 72 434 V HG2 H 1 0.725 1 73 434 V N N 15
114.74 1 74 435 F CA C 13 55.321 1 75 435 F CB C 13 37.701 1 76 435
F CO C 13 177.18 1 77 435 F H H 1 7.344 1 78 435 F N N 15 117.562 1
79 436 R CA C 13 56.384 1 80 436 R CB C 13 30.009 1 81 436 R CO C
13 176.099 1 82 436 R H H 1 7.225 1 83 436 R N N 15 118.73 1 84 437
N CA C 13 53.605 1 85 437 N CB C 13 38.193 1 86 437 N H H 1 8.252 1
87 437 N N N 15 119.685 1 88 438 G CA C 13 44.807 1 89 438 G CO C
13 172.725 1 90 438 G H H 1 8.08 1 91 438 G N N 15 109.481 1 92 439
N CA C 13 52.847 1 93 439 N CB C 13 37.175 1 94 439 N H H 1 8.737 1
95 439 N N N 15 120.443 1 96 440 Q CA C 13 58.04 1 97 440 Q CB C 13
28.495 1 98 440 Q CO C 13 175.605 1 99 440 Q H H 1 9.022 1 100 440
Q N N 15 125.59 1 101 441 D CA C 13 53.428 1 102 441 D CB C 13
40.409 1 103 441 D CO C 13 175.45 1 104 441 D H H 1 7.969 1 105 441
D N N 15 114.845 1 106 442 E CA C 13 56.501 1 107 442 E CB C 13
30.044 1 108 442 E CO C 13 175.905 1 109 442 E H H 1 7.143 1 110
442 E N N 15 121.501 1 111 443 V CA C 13 64.02 1 112 443 V CB C 13
31.155 1 113 443 V CG1 C 13 21.487 1 114 443 V CG2 C 13 21.844 1
115 443 V H H 1 8.65 1 116 443 V HG1 H 1 0.721 1 117 443 V HG2 H 1
0.882 1 118 443 V N N 15 127.026 1 119 444 L CA C 13 54.051 1 120
444 L CB C 13 43.293 1 121 444 L CD1 C 13 27.029 1 122 444 L CD2 C
13 21.807 1 123 444 L CO C 13 176.79 1 124 444 L H H 1 9.012 1 125
444 L HD1 H 1 0.59 1 126 444 L HD2 H 1 0.597 1 127 444 L N N 15
127.296 1 128 445 S CA C 13 57.432 1 129 445 S CB C 13 64.093 1 130
445 S CO C 13 172 1 131 445 S H H 1 7.412 1 132 445 S N N 15
111.726 1 133 446 E CA C 13 55.317 1 134 446 E CB C 13 31.965 1 135
446 E CO C 13 174.257 1 136 446 E H H 1 7.933 1 137 446 E N N 15
125.063 1 138 447 A CA C 13 51.875 1 139 447 A CB C 13 18.979 1 140
447 A CO C 13 176.087 1 141 447 A H H 1 8.286 1 142 447 A N N 15
124.213 1 143 448 F CA C 13 56.316 1 144 448 F CB C 13 36.029 1 145
448 F CO C 13 175.788 1 146 448 F H H 1 8.61 1 147 448 F N N 15
115.367 1 148 449 R CA C 13 57.675 1 149 449 R CB C 13 26.229 1 150
449 R CO C 13 175.491 1 151 449 R H H 1 8.484 1 152 449 R N N 15
110.844 1 153 450 L CA C 13 53.763 1 154 450 L CB C 13 44.035 1 155
450 L CD1 C 13 25.813 1 156 450 L CD2 C 13 22.437 1 157 450 L CO C
13 176.645 1 158 450 L H H 1 8.389 1 159 450 L HD1 H 1 0.89 1 160
450 L HD2 H 1 0.948 1 161 450 L N N 15 121.623 1 162 451 T CA C 13
60.834 1 163 451 T CB C 13 71.178 1 164 451 T CO C 13 173.407 1 165
451 T H H 1 8.315 1 166 451 T N N 15 113.296 1 167 452 I CA C 13
56.522 1 168 452 I CB C 13 36.082 1 169 452 I CO C 13 176.082 1 170
452 I H H 1 8.521 1 171 452 I N N 15 124.173 1 172 453 T CA C 13
59.709 1 173 453 T CB C 13 72.939 1 174 453 T H H 1 9.811 1 175 453
T N N 15 119.807 1 176 454 R CA C 13 60.137 1 177 454 R CB C 13
28.989 1 178 454 R CO C 13 177.392 1 179 454 R H H 1 8.211 1 180
454 R N N 15 122.061 1 181 455 K CA C 13 59.289 1 182 455 K CB C 13
31.114 1 183 455 K CO C 13 178.628 1 184 455 K H H 1 8.504 1 185
455 K N N 15 119.122 1 186 456 D CA C 13 57.369 1 187 456 D CB C 13
40.809 1 188 456 D H H 1 7.271 1 189 456 D N N 15 117.779 1 190 457
I CA C 13 62.392 1 191 457 I CB C 13 37.06 1 192 457 I H H 1 8.159
1 193 457 I N N 15 121.588 1 194 458 Q CA C 13 57.804 1 195 458 Q
CB C 13 26.567 1 196 458 Q CO C 13 178.732 1 197 458 Q H H 1 7.923
1 198 458 Q N N 15 117.897 1 199 459 T CA C 13 65.051 1 200 459 T
CB C 13 67.395 1 201 459 T H H 1 7.897 1 202 459 T N N 15 113.263 1
203 460 L CA C 13 54.923 1 204 460 L CB C 13 41.723 1 205 460 L CD1
C 13 25.968 1 206 460 L CD2 C 13 25.889 1 207 460 L CO C 13 179.644
1 208 460 L H H 1 7.253 1 209 460 L HD1 H 1 0.82 1 210 460 L HD2 H
1 0.925 1 211 460 L N N 15 115.083 1 212 461 N CA C 13 51.888 1 213
461 N CB C 13 37.194 1 214 461 N H H 1 7.421 1
215 461 N N N 15 119.845 1 216 462 H CA C 13 57.014 1 217 462 H CB
C 13 28.992 1 218 462 H H H 1 7.773 1 219 462 H N N 15 119.821 1
220 465 W CA C 13 56.901 1 221 465 W CB C 13 27.801 1 222 465 W H H
1 8.319 1 223 465 W HE1 H 1 10.206 1 224 465 W N N 15 120.321 1 225
465 W NE1 N 15 130.435 1 226 466 L CA C 13 57.74 1 227 466 L CB C
13 41.916 1 228 466 L CD1 C 13 25.446 1 229 466 L CD2 C 13 23.298 1
230 466 L H H 1 7.644 1 231 466 L HD1 H 1 0.634 1 232 466 L HD2 H 1
0.563 1 233 466 L N N 15 125.508 1 234 467 N CA C 13 50.295 1 235
467 N CB C 13 39.556 1 236 467 N CO C 13 174.619 1 237 467 N H H 1
7.164 1 238 467 N N N 15 116.901 1 239 468 D CA C 13 57.481 1 240
468 D CB C 13 40.531 1 241 468 D H H 1 8.246 1 242 468 D N N 15
115.434 1 243 469 E CA C 13 60.578 1 244 469 E CB C 13 27.414 1 245
469 E CO C 13 179.948 1 246 469 E H H 1 8.991 1 247 469 E N N 15
119.089 1 248 470 I CA C 13 61.231 1 249 470 I CB C 13 34.744 1 250
470 I CO C 13 177.04 1 251 470 I H H 1 7.753 1 252 470 I N N 15
117.845 1 253 471 I CA C 13 64.903 1 254 471 I H H 1 6.974 1 255
471 I N N 15 117.941 1 256 472 N CA C 13 56.07 1 257 472 N CB C 13
37.624 1 258 472 N CO C 13 178.288 1 259 472 N H H 1 9.03 1 260 472
N N N 15 115.254 1 261 473 F CA C 13 62.584 1 262 473 F CB C 13
39.667 1 263 473 F CO C 13 177.436 1 264 473 F H H 1 8.304 1 265
473 F N N 15 123.63 1 266 474 Y CA C 13 62.784 1 267 474 Y CB C 13
38.335 1 268 474 Y CO C 13 178.151 1 269 474 Y H H 1 8.774 1 270
474 Y N N 15 120.467 1 271 475 M CA C 13 57.325 1 272 475 M CB C 13
31.041 1 273 475 M CO C 13 179.367 1 274 475 M H H 1 8.709 1 275
475 M N N 15 115.346 1 276 476 N CA C 13 56.292 1 277 476 N CB C 13
37.912 1 278 476 N CO C 13 177.604 1 279 476 N H H 1 7.371 1 280
476 N N N 15 117.074 1 281 477 M CA C 13 59.952 1 282 477 M CB C 13
31.504 1 283 477 M CO C 13 179.545 1 284 477 M H H 1 7.664 1 285
477 M N N 15 121.465 1 286 478 L CA C 13 57.471 1 287 478 L CB C 13
39.79 1 288 478 L CD1 C 13 27.34 1 289 478 L CD2 C 13 22.112 1 290
478 L CO C 13 180.857 1 291 478 L H H 1 7.767 1 292 478 L HD1 H 1
0.658 1 293 478 L HD2 H 1 0.411 1 294 478 L N N 15 119.925 1 295
479 M CA C 13 59.413 1 296 479 M CB C 13 32.433 1 297 479 M CO C 13
179.134 1 298 479 M H H 1 7.603 1 299 479 M N N 15 118.957 1 300
480 E CA C 13 59.225 1 301 480 E CB C 13 28.37 1 302 480 E H H 1
8.059 1 303 480 E N N 15 122.932 1 304 481 R CA C 13 58.251 1 305
481 R CB C 13 28.749 1 306 481 R CO C 13 176.421 1 307 481 R H H 1
7.917 1 308 481 R N N 15 120.662 1 309 482 S CA C 13 60.255 1 310
482 S CB C 13 63.102 1 311 482 S CO C 13 172.394 1 312 482 S H H 1
7.201 1 313 482 S N N 15 113.273 1 314 483 K CA C 13 56.793 1 315
483 K CB C 13 31.687 1 316 483 K CO C 13 178.176 1 317 483 K H H 1
6.968 1 318 483 K N N 15 118.755 1 319 484 E CB C 13 29.049 1 320
484 E CO C 13 176.653 1 321 484 E H H 1 8.114 1 322 484 E N N 15
121.011 1 323 485 K CA C 13 57.55 1 324 485 K CB C 13 31.154 1 325
485 K CO C 13 177.924 1 326 485 K H H 1 8.263 1 327 485 K N N 15
121.725 1 328 486 G CA C 13 44.731 1 329 486 G CO C 13 173.993 1
330 486 G H H 1 8.738 1 331 486 G N N 15 111.446 1 332 487 L CA C
13 52.224 1 333 487 L CB C 13 40.075 1 334 487 L CD1 C 13 25.797 1
335 487 L CD2 C 13 23.228 1 336 487 L CO C 13 174.966 1 337 487 L H
H 1 7.357 1 338 487 L HD1 H 1 0.778 1 339 487 L HD2 H 1 0.829 1 340
487 L N N 15 121.648 1 341 489 S CA C 13 57.732 1 342 489 S CB C 13
63.976 1 343 489 S CO C 13 175.307 1 344 489 S H H 1 9.146 1 345
489 S N N 15 117.954 1 346 490 V CA C 13 59.96 1 347 490 V CB C 13
36.725 1 348 490 V CG1 C 13 21.034 1 349 490 V CG2 C 13 23.035 1
350 490 V CO C 13 175.445 1 351 490 V H H 1 7.378 1 352 490 V HG1 H
1 0.555 1 353 490 V HG2 H 1 0.885 1 354 490 V N N 15 118.616 1 355
491 H CA C 13 56.457 1 356 491 H CB C 13 33.175 1 357 491 H CO C 13
172.689 1 358 491 H H H 1 8.824 1 359 491 H N N 15 124.16 1 360 492
A CA C 13 48.933 1 361 492 A CB C 13 20.637 1 362 492 A CO C 13
175.149 1 363 492 A H H 1 7.475 1 364 492 A N N 15 129.587 1 365
493 F CA C 13 57.443 1 366 493 F CB C 13 40.032 1 367 493 F H H 1
8.075 1 368 493 F N N 15 120.292 1 369 494 N CA C 13 52.612 1 370
494 N CB C 13 39.014 1 371 494 N CO C 13 177.042 1 372 494 N H H 1
8.614 1 373 494 N N N 15 116.324 1 374 495 T CA C 13 65.108 1 375
495 T CB C 13 67.954 1 376 495 T H H 1 8.712 1 377 495 T N N 15
111.881 1 378 496 F CA C 13 57.589 1 379 496 F CB C 13 38.722 1 380
496 F CO C 13 176.791 1 381 496 F H H 1 8.441 1 382 496 F N N 15
120.392 1 383 497 F CA C 13 61.468 1 384 497 F CB C 13 38.442 1 385
497 F CO C 13 175.62 1 386 497 F H H 1 7.951 1 387 497 F N N 15
121.386 1 388 498 F CA C 13 62.45 1 389 498 F CB C 13 37.649 1 390
498 F CO C 13 176.151 1 391 498 F H H 1 10.059 1 392 498 F N N 15
120.473 1 393 499 T CA C 13 65.751 1 394 499 T CB C 13 68.656 1 395
499 T H H 1 7.099 1 396 499 T N N 15 111.797 1 397 500 K CA C 13
58.082 1 398 500 K CB C 13 30.293 1 399 500 K CO C 13 177.17 1 400
500 K H H 1 7.805 1 401 500 K N N 15 122.907 1 402 501 L CA C 13
56.922 1 403 501 L CB C 13 40.32 1 404 501 L CD1 C 13 21.344 1 405
501 L CD2 C 13 26.13 1 406 501 L H H 1 8.04 1 407 501 L HD1 H 1
0.619 1 408 501 L HD2 H 1 0.269 1 409 501 L N N 15 120.722 1 410
502 K CA C 13 58.359 1 411 502 K CB C 13 31.117 1 412 502 K CO C 13
177.542 1 413 502 K H H 1 8.113 1 414 502 K N N 15 117.113 1 415
503 T CA C 13 63.65 1 416 503 T CB C 13 69.641 1 417 503 T CO C 13
175.362 1 418 503 T H H 1 7.521 1 419 503 T N N 15 108.626 1 420
504 A CA C 13 51.681 1 421 504 A CB C 13 19.982 1 422 504 A CO C 13
177.923 1 423 504 A H H 1 8.417 1 424 504 A N N 15 124.229 1 425
505 G CA C 13 44.062 1 426 505 G CO C 13 173.703 1 427 505 G H H 1
7.404 1 428 505 G N N 15 108.216 1 429 506 Y CA C 13 61.372 1 430
506 Y CB C 13 38.185 1 431 506 Y CO C 13 177.707 1 432 506 Y H H 1
8.506 1 433 506 Y N N 15 118.015 1 434 507 Q CA C 13 58.073 1 435
507 Q CB C 13 26.321 1 436 507 Q CO C 13 177.318 1 437 507 Q H H 1
8.677 1 438 507 Q N N 15 113.949 1 439 508 A CA C 13 53.059 1 440
508 A CB C 13 19.41 1 441 508 A CO C 13 178.474 1 442 508 A H H 1
7.193 1 443 508 A N N 15 117.81 1 444 509 V CA C 13 59.584 1 445
509 V CB C 13 32.636 1 446 509 V CG1 C 13 19.036 1 447 509 V CG2 C
13 20.077 1 448 509 V CO C 13 178.833 1 449 509 V H H 1 6.99 1 450
509 V HG1 H 1 0.152 1 451 509 V HG2 H 1 0.505 1 452 509 V N N 15
104.928 1 453 510 K CA C 13 59.235 1 454 510 K CB C 13 30.396 1 455
510 K CO C 13 178.002 1 456 510 K H H 1 7.252 1 457 510 K N N 15
126.565 1 458 511 R CA C 13 56.969 1 459 511 R CB C 13 28.393 1 460
511 R H H 1 8.593 1 461 511 R N N 15 116.236 1 462 512 W CA C 13
59.154 1 463 512 W CB C 13 27.825 1 464 512 W CO C 13 178.179 1 465
512 W H H 1 8.477 1
466 512 W HE1 H 1 10.293 1 467 512 W N N 15 120.092 1 468 512 W NE1
N 15 129.338 1 469 513 T CA C 13 60 1 470 513 T CB C 13 65.562 1
471 513 T CO C 13 174.181 1 472 513 T H H 1 7.356 1 473 513 T N N
15 105.836 1 474 514 K CA C 13 59.285 1 475 514 K CB C 13 31.488 1
476 514 K CO C 13 177.271 1 477 514 K H H 1 7.187 1 478 514 K N N
15 120.77 1 479 515 K CA C 13 55.075 1 480 515 K CB C 13 31.34 1
481 515 K CO C 13 175.55 1 482 515 K H H 1 8.52 1 483 515 K N N 15
115.267 1 484 516 V CA C 13 60.315 1 485 516 V CB C 13 34.794 1 486
516 V CG1 C 13 22.213 1 487 516 V CG2 C 13 19.431 1 488 516 V CO C
13 173.373 1 489 516 V H H 1 7.346 1 490 516 V HG1 H 1 1.035 1 491
516 V HG2 H 1 0.828 1 492 516 V N N 15 118.521 1 493 517 D CA C 13
50.719 1 494 517 D CB C 13 39.298 1 495 517 D CO C 13 178.171 1 496
517 D H H 1 8.502 1 497 517 D N N 15 124.325 1 498 518 V CA C 13
64.12 1 499 518 V CB C 13 30.53 1 500 518 V CG1 C 13 21.974 1 501
518 V CG2 C 13 17.74 1 502 518 V CO C 13 173.205 1 503 518 V H H 1
8.909 1 504 518 V HG1 H 1 0.709 1 505 518 V HG2 H 1 0.246 1 506 518
V N N 15 121.419 1 507 519 F CA C 13 57.9 1 508 519 F CB C 13
36.872 1 509 519 F CO C 13 176.5 1 510 519 F H H 1 7.223 1 511 519
F N N 15 110.893 1 512 520 S CB C 13 64.082 1 513 520 S CO C 13
173.635 1 514 520 S H H 1 7.457 1 515 520 S N N 15 113.527 1 516
521 V CA C 13 58.421 1 517 521 V CB C 13 33.049 1 518 521 V CG1 C
13 21.474 1 519 521 V CG2 C 13 19.203 1 520 521 V CO C 13 174.363 1
521 521 V H H 1 6.675 1 522 521 V HG1 H 1 0.677 1 523 521 V HG2 H 1
0.736 1 524 521 V N N 15 114.244 1 525 522 D CA C 13 57.671 1 526
522 D CB C 13 42.241 1 527 522 D H H 1 8.177 1 528 522 D N N 15
120.102 1 529 523 I CA C 13 59.234 1 530 523 I H H 1 8.209 1 531
523 I N N 15 117.31 1 532 524 L CA C 13 51.912 1 533 524 L CB C 13
42.117 1 534 524 L CD1 C 13 24.261 2 535 524 L CD2 C 13 24.458 2
536 524 L H H 1 9.357 1 537 524 L HD1 H 1 0.826 2 538 524 L HD2 H 1
0.873 2 539 524 L N N 15 121.905 1 540 525 L CA C 13 53.109 1 541
525 L CB C 13 43.667 1 542 525 L CD1 C 13 27.473 1 543 525 L CD2 C
13 23.613 1 544 525 L H H 1 8.708 1 545 525 L HD1 H 1 0.737 1 546
525 L HD2 H 1 0.713 1 547 525 L N N 15 120.45 1 548 526 V CA C 13
59.564 1 549 526 V CB C 13 32.337 1 550 526 V CG1 C 13 20.681 1 551
526 V CG2 C 13 19.401 1 552 526 V H H 1 8.925 1 553 526 V HG1 H 1
-0.236 1 554 526 V HG2 H 1 0.488 1 555 526 V N N 15 120.847 1 556
528 I CA C 13 60.95 1 557 528 I CB C 13 39.801 1 558 528 I H H 1
8.737 1 559 528 I N N 15 125.023 1 560 529 H CA C 13 50.979 1 561
529 H CB C 13 29.375 1 562 529 H CO C 13 174.067 1 563 529 H H H 1
9.036 1 564 529 H N N 15 129.849 1 565 530 L CA C 13 52.799 1 566
530 L CB C 13 41.01 1 567 530 L CD1 C 13 25.841 1 568 530 L CD2 C
13 23.767 1 569 530 L CO C 13 176.318 1 570 530 L H H 1 8.525 1 571
530 L HD1 H 1 0.874 1 572 530 L HD2 H 1 0.77 1 573 530 L N N 15
130.501 1 574 531 G CA C 13 46.001 1 575 531 G CO C 13 174.79 1 576
531 G H H 1 8.157 1 577 531 G N N 15 115.336 1 578 532 V CA C 13
61.094 1 579 532 V CB C 13 30.784 1 580 532 V CG1 C 13 20.964 1 581
532 V CG2 C 13 18.117 1 582 532 V CO C 13 175.461 1 583 532 V H H 1
8.198 1 584 532 V HG1 H 1 0.532 1 585 532 V HG2 H 1 0.584 1 586 532
V N N 15 119.81 1 587 533 H CA C 13 55.28 1 588 533 H CB C 13
32.996 1 589 533 H CO C 13 174.498 1 590 533 H H H 1 7.803 1 591
533 H N N 15 121.771 1 592 534 W CA C 13 55.915 1 593 534 W CB C 13
32.49 1 594 534 W H H 1 6.407 1 595 534 W HE1 H 1 9.377 1 596 534 W
N N 15 125.3 1 597 534 W NE1 N 15 128.192 1 598 535 C CA C 13
56.548 1 599 535 C CB C 13 30.615 1 600 535 C CO C 13 171.965 1 601
535 C H H 1 9.461 1 602 535 C N N 15 117.22 1 603 536 L CA C 13
54.008 1 604 536 L CB C 13 46.487 1 605 536 L CD1 C 13 22.301 1 606
536 L CD2 C 13 26.282 1 607 536 L H H 1 7.905 1 608 536 L HD1 H 1
0.679 1 609 536 L HD2 H 1 0.597 1 610 536 L N N 15 120.825 1 611
537 A CA C 13 49.576 1 612 537 A CB C 13 20.964 1 613 537 A H H 1
8.835 1 614 537 A N N 15 126.773 1 615 538 V CA C 13 60.449 1 616
538 V CB C 13 35.413 1 617 538 V CG1 C 13 21.698 1 618 538 V CG2 C
13 21.913 1 619 538 V CO C 13 174.727 1 620 538 V H H 1 9.071 1 621
538 V HG1 H 1 0.87 1 622 538 V HG2 H 1 0.809 1 623 538 V N N 15
119.546 1 624 539 V CA C 13 60.873 1 625 539 V CB C 13 32.053 1 626
539 V CG1 C 13 20.502 1 627 539 V CG2 C 13 19.475 1 628 539 V H H 1
9.402 1 629 539 V HG1 H 1 0.441 1 630 539 V HG2 H 1 0.881 1 631 539
V N N 15 130.501 1 632 540 D CA C 13 51.922 1 633 540 D CB C 13
41.816 1 634 540 D H H 1 8.954 1 635 540 D N N 15 126.546 1 636 541
F CA C 13 62.022 1 637 541 F CB C 13 39.187 1 638 541 F H H 1 9.479
1 639 541 F N N 15 123.936 1 640 542 R CA C 13 57.3 1 641 542 R CB
C 13 28.607 1 642 542 R CO C 13 179.074 1 643 542 R H H 1 8.714 1
644 542 R N N 15 117.561 1 645 543 K CA C 13 54.808 1 646 543 K CB
C 13 33.288 1 647 543 K CO C 13 175.221 1 648 543 K H H 1 6.749 1
649 543 K N N 15 114.224 1 650 544 K CA C 13 55.562 1 651 544 K CB
C 13 27.641 1 652 544 K CO C 13 175.247 1 653 544 K H H 1 7.423 1
654 544 K N N 15 115.776 1 655 545 N CA C 13 51.023 1 656 545 N CB
C 13 42.123 1 657 545 N CO C 13 173.859 1 658 545 N H H 1 7.23 1
659 545 N N N 15 113.498 1 660 546 I CA C 13 61.517 1 661 546 I H H
1 8.432 1 662 546 I N N 15 120.288 1 663 547 T CA C 13 60.921 1 664
547 T CB C 13 70.493 1 665 547 T H H 1 8.781 1 666 547 T N N 15
121.352 1 667 548 Y CA C 13 57.227 1 668 548 Y CB C 13 40.796 1 669
548 Y H H 1 8.727 1 670 548 Y N N 15 128.981 1 671 549 Y CB C 13
40.072 1 672 549 Y H H 1 9.079 1 673 549 Y N N 15 125.239 1 674 550
D CA C 13 52.549 1 675 550 D CB C 13 43.937 1 676 550 D CO C 13
177.444 1 677 550 D H H 1 8.116 1 678 550 D N N 15 123.169 1 679
551 S CA C 13 60.463 1 680 551 S CB C 13 62.962 1 681 551 S CO C 13
174.422 1 682 551 S H H 1 9.519 1 683 551 S N N 15 122.969 1 684
552 M CA C 13 54.901 1 685 552 M CB C 13 34.489 1 686 552 M CO C 13
178.972 1 687 552 M H H 1 9.32 1 688 552 M N N 15 122.574 1 689 553
G CA C 13 46.475 1 690 553 G CO C 13 175.385 1 691 553 G H H 1 7.89
1 692 553 G N N 15 109.507 1 693 554 G CA C 13 44.928 1 694 554 G
CO C 13 171.658 1 695 554 G H H 1 7.51 1 696 554 G N N 15 107.555 1
697 555 I CA C 13 59.215 1 698 555 I CB C 13 37.955 1 699 555 I CO
C 13 176.285 1 700 555 I H H 1 8.05 1 701 555 I N N 15 118.138 1
702 556 N CA C 13 50.688 1 703 556 N CB C 13 36.209 1 704 556 N H H
1 7.762 1 705 556 N N N 15 124.106 1 706 557 N CA C 13 55.66 1 707
557 N CB C 13 37.225 1 708 557 N H H 1 8.339 1 709 557 N N N 15
121.312 1 710 558 E CA C 13 59.21 1 711 558 E CB C 13 28.223 1 712
558 E H H 1 8.531 1 713 558 E N N 15 120.721 1 714 559 A CA C 13
55.072 1 715 559 A CB C 13 17.209 1 716 559 A CO C 13 179.228 1
717 559 A H H 1 7.491 1 718 559 A N N 15 120.499 1 719 560 C CA C
13 61.861 1 720 560 C CB C 13 26.362 1 721 560 C CO C 13 176.152 1
722 560 C H H 1 6.803 1 723 560 C N N 15 111.946 1 724 561 R CA C
13 59.736 1 725 561 R CB C 13 29.122 1 726 561 R CO C 13 179.458 1
727 561 R H H 1 8.077 1 728 561 R N N 15 120.238 1 729 562 I CA C
13 64.773 1 730 562 I CB C 13 36.965 1 731 562 I CO C 13 179.283 1
732 562 I H H 1 8.583 1 733 562 I N N 15 120.588 1 734 563 L CA C
13 56.989 1 735 563 L CB C 13 41.139 1 736 563 L CD1 C 13 26.07 1
737 563 L CD2 C 13 22.794 1 738 563 L CO C 13 177.668 1 739 563 L H
H 1 7.58 1 740 563 L HD1 H 1 0.714 1 741 563 L HD2 H 1 0.791 1 742
563 L N N 15 120.645 1 743 564 L CA C 13 57.863 1 744 564 L CB C 13
40.586 1 745 564 L CD1 C 13 23.029 1 746 564 L CD2 C 13 25.722 1
747 564 L H H 1 7.989 1 748 564 L HD1 H 1 0.503 1 749 564 L HD2 H 1
0.873 1 750 564 L N N 15 122.277 1 751 565 Q CB C 13 27.023 1 752
565 Q CO C 13 178.554 1 753 565 Q H H 1 7.945 1 754 565 Q N N 15
116.169 1 755 566 Y CA C 13 61.262 1 756 566 Y CB C 13 36.89 1 757
566 Y H H 1 8.147 1 758 566 Y N N 15 121.414 1 759 567 L CA C 13
57.69 1 760 567 L CB C 13 39.693 1 761 567 L CD1 C 13 26.027 1 762
567 L CD2 C 13 21.698 1 763 567 L H H 1 7.756 1 764 567 L HD1 H 1
0.298 1 765 567 L HD2 H 1 0.574 1 766 567 L N N 15 118.927 1 767
568 K CA C 13 59.666 1 768 568 K CB C 13 31.072 1 769 568 K CO C 13
180.159 1 770 568 K H H 1 7.436 1 771 568 K N N 15 116.321 1 112
569 Q CA C 13 58.376 1 773 569 Q CB C 13 26.837 1 774 569 Q CO C 13
178.121 1 775 569 Q H H 1 7.71 1 776 569 Q N N 15 119.281 1 111 570
E CA C 13 57.248 1 778 570 E CB C 13 27.85 1 779 570 E CO C 13
178.511 1 780 570 E H H 1 8.874 1 781 570 E N N 15 123.757 1 782
571 S CA C 13 61.824 1 783 571 S CB C 13 63.591 1 784 571 S H H 1
8.112 1 785 571 S N N 15 113.072 1 786 572 I CA C 13 64.046 1 787
572 I CB C 13 36.762 1 788 572 I CO C 13 178.898 1 789 572 I H H 1
7.085 1 790 572 I N N 15 120.025 1 791 573 D CA C 13 58.589 1 792
573 D CB C 13 45.295 1 793 573 D H H 1 8.267 1 794 573 D N N 15
119.596 1 795 574 K CA C 13 55.261 1 796 574 K H H 1 8.537 1 797
574 K N N 15 110.111 1 798 575 K CA C 13 53.6 1 799 575 K H H 1
7.814 1 800 575 K N N 15 114.921 1 801 580 D CA C 13 53.108 1 802
580 D CO C 13 175.974 1 803 580 D H H 1 8.008 1 804 580 D N N 15
128.24 1 805 581 T CA C 13 61.607 1 806 581 T CB C 13 67.992 1 807
581 T CO C 13 176.37 1 808 581 T H H 1 8.005 1 809 581 T N N 15
114.488 1 810 582 N CA C 13 55.671 1 811 582 N CB C 13 37.704 1 812
582 N CO C 13 177.026 1 813 582 N H H 1 8.559 1 814 582 N N N 15
124.721 1 815 583 G CA C 13 45.005 1 816 583 G CO C 13 174.64 1 817
583 G H H 1 8.964 1 818 583 G N N 15 113.066 1 819 584 W CA C 13
58.735 1 820 584 W CB C 13 27.343 1 821 584 W CO C 13 177.331 1 822
584 W H H 1 7.891 1 823 584 W HE1 H 1 10.207 1 824 584 W N N 15
120.551 1 825 584 W NE1 N 15 130.072 1 826 585 Q CA C 13 54.336 1
827 585 Q CB C 13 32.617 1 828 585 Q CO C 13 173.396 1 829 585 Q H
H 1 8.32 1 830 585 Q N N 15 120.47 1 831 586 L CA C 13 52.787 1 832
586 L CB C 13 41.572 1 833 586 L CD1 C 13 24.58 1 834 586 L CD2 C
13 24.177 1 835 586 L CO C 13 176.073 1 836 586 L H H 1 8.257 1 837
586 L HD1 H 1 0.875 1 838 586 L HD2 H 1 1.06 1 839 586 L N N 15
122.471 1 840 587 F CA C 13 56.274 1 841 587 F CB C 13 41.98 1 842
587 F CO C 13 174.71 1 843 587 F H H 1 9.007 1 844 587 F N N 15
119.628 1 845 588 S CA C 13 57.584 1 846 588 S CB C 13 64.741 1 847
588 S CO C 13 174.522 1 848 588 S H H 1 8.55 1 849 588 S N N 15
115.508 1 850 589 K CA C 13 54.504 1 851 589 K CB C 13 30.677 1 852
589 K CO C 13 176.928 1 853 589 K H H 1 8.375 1 854 589 K N N 15
123.879 1 855 590 K CA C 13 55.59 1 856 590 K CB C 13 32.654 1 857
590 K CO C 13 178.646 1 858 590 K H H 1 9.16 1 859 590 K N N 15
124.442 1 860 591 S CA C 13 60.73 1 861 591 S CB C 13 62.469 1 862
591 S CO C 13 175.07 1 863 591 S H H 1 8.771 1 864 591 S N N 15
116.878 1 865 592 Q CA C 13 56.481 1 866 592 Q CB C 13 27.319 1 867
592 Q CO C 13 177.119 1 868 592 Q H H 1 7.787 1 869 592 Q N N 15
114.421 1 870 593 E CA C 13 56.788 1 871 593 E CB C 13 31.514 1 872
593 E CO C 13 176.185 1 873 593 E H H 1 8.147 1 874 593 E N N 15
116.571 1 875 594 I CA C 13 57.233 1 876 594 I CB C 13 39.464 1 877
594 I H H 1 7.099 1 878 594 I N N 15 111.638 1 879 596 Q CA C 13
52.641 1 880 596 Q CB C 13 31.367 1 881 596 Q CO C 13 176.998 1 882
596 Q H H 1 8.568 1 883 596 Q N N 15 119.776 1 884 597 Q CA C 13
53.866 1 885 597 Q CB C 13 28.195 1 886 597 Q CO C 13 175.677 1 887
597 Q H H 1 8.67 1 888 597 Q N N 15 118.265 1 889 598 M CA C 13
55.496 1 890 598 M CB C 13 33.978 1 891 598 M CO C 13 175.887 1 892
598 M H H 1 9.45 1 893 598 M N N 15 118.496 1 894 599 N CA C 13
51.768 1 895 599 N CB C 13 39.114 1 896 599 N H H 1 7.565 1 897 599
N N N 15 117.184 1 898 600 G H H 1 9.054 1 899 600 G N N 15 114.081
1 900 601 S CA C 13 58.424 1 901 601 S CB C 13 60.647 1 902 601 S H
H 1 7.855 1 903 601 S N N 15 114.866 1 904 602 D CA C 13 55.181 1
905 602 D CB C 13 40.81 1 906 602 D CO C 13 178.539 1 907 602 D H H
1 7.257 1 908 602 D N N 15 118.282 1 909 603 C CA C 13 60.678 1 910
603 C CB C 13 28.27 1 911 603 C CO C 13 175.783 1 912 603 C H H 1
7.715 1 913 603 C N N 15 121.968 1 914 604 G CA C 13 46.862 1 915
604 G CO C 13 175.076 1 916 604 G H H 1 8.736 1 917 604 G N N 15
109.643 1 918 605 M CA C 13 54.421 1 919 605 M CB C 13 28.885 1 920
605 M CO C 13 178.831 1 921 605 M H H 1 6.973 1 922 605 M N N 15
118.381 1 923 606 F CA C 13 63.186 1 924 606 F CB C 13 37.346 1 925
606 F CO C 13 175.928 1 926 606 F H H 1 8.265 1 927 606 F N N 15
118.962 1 928 607 A CA C 13 55.826 1 929 607 A CB C 13 15.841 1 930
607 A CO C 13 179.705 1 931 607 A H H 1 7.745 1 932 607 A N N 15
118.262 1 933 608 C CA C 13 64.555 1 934 608 C CB C 13 26.489 1 935
608 C CO C 13 176.344 1 936 608 C H H 1 7.15 1 937 608 C N N 15
111.327 1 938 609 K CA C 13 55.983 1 939 609 K CB C 13 28.003 1 940
609 K CO C 13 180.898 1 941 609 K H H 1 8.114 1 942 609 K N N 15
117.546 1 943 610 Y CA C 13 58.081 1 944 610 Y CB C 13 36.268 1 945
610 Y CO C 13 177.942 1 946 610 Y H H 1 9.584 1 947 610 Y N N 15
121.342 1 948 611 A CA C 13 55.284 1 949 611 A CB C 13 17.36 1 950
611 A CO C 13 179.296 1 951 611 A H H 1 7.3 1 952 611 A N N 15
118.235 1 953 612 D CA C 13 57.496 1 954 612 D CB C 13 40.809 1 955
612 D CO C 13 177.083 1 956 612 D H H 1 8.285 1 957 612 D N N 15
119.051 1 958 613 C CA C 13 64.249 1 959 613 C CB C 13 26.401 1 960
613 C CO C 13 177.033 1 961 613 C H H 1 7.283 1 962 613 C N N 15
114.15 1 963 614 I CA C 13 64.188 1 964 614 I CB C 13 38.287 1 965
614 I CO C 13 180.531 1 966 614 I H H 1 8.523 1 967 614 I N N 15
119.103 1
968 615 T CB C 13 67.658 1 969 615 T CO C 13 174.525 1 970 615 T H
H 1 8.251 1 971 615 T N N 15 108.325 1 972 616 K CA C 13 55.419 1
973 616 K CB C 13 31.971 1 974 616 K CO C 13 175.509 1 975 616 K H
H 1 7.204 1 976 616 K N N 15 118.784 1 977 617 D CA C 13 55.028 1
978 617 D CB C 13 38.871 1 979 617 D CO C 13 174.882 1 980 617 D H
H 1 7.982 1 981 617 D N N 15 117.696 1 982 618 R CA C 13 51.906 1
983 618 R CB C 13 30.614 1 984 618 R CO C 13 173.638 1 985 618 R H
H 1 7.893 1 986 618 R N N 15 116.707 1 987 620 I CA C 13 62.112 1
988 620 I CB C 13 35.731 1 989 620 I CO C 13 177.018 1 990 620 I H
H 1 8.465 1 991 620 I N N 15 121.915 1 992 621 N CA C 13 52.404 1
993 621 N CB C 13 38.108 1 994 621 N H H 1 7.952 1 995 621 N N N 15
126.058 1 996 622 F CA C 13 54.399 1 997 622 F CB C 13 41.066 1 998
622 F CO C 13 173.442 1 999 622 F H H 1 6.573 1 1000 622 F N N 15
114.01 1 1001 623 T CA C 13 59.922 1 1002 623 T CB C 13 73.255 1
1003 623 T H H 1 11.019 1 1004 623 T N N 15 112.666 1 1005 624 Q CA
C 13 57.991 1 1006 624 Q CB C 13 28.115 1 1007 624 Q CO C 13
177.817 1 1008 624 Q H H 1 9.841 1 1009 624 Q N N 15 118.693 1 1010
625 Q CA C 13 57.772 1 1011 625 Q CB C 13 27.232 1 1012 625 Q CO C
13 177.118 1 1013 625 Q H H 1 8.35 1 1014 625 Q N N 15 118.528 1
1015 626 H CA C 13 59.023 1 1016 626 H CB C 13 32.018 1 1017 626 H
CO C 13 175.195 1 1018 626 H H H 1 7.69 1 1019 626 H N N 15 116.12
1 1020 627 M CA C 13 58.703 1 1021 627 M CB C 13 29.282 1 1022 627
M CO C 13 175.481 1 1023 627 M H H 1 7.581 1 1024 627 M N N 15
117.619 1 1025 629 Y CA C 13 59.898 1 1026 629 Y CB C 13 36.935 1
1027 629 Y CO C 13 176.303 1 1028 629 Y H H 1 7.455 1 1029 629 Y N
N 15 119.231 1 1030 630 F CA C 13 57.548 1 1031 630 F CO C 13
179.707 1 1032 630 F H H 1 8.72 1 1033 630 F N N 15 118.661 1 1034
631 R CA C 13 59.879 1 1035 631 R CB C 13 29.774 1 1036 631 R CO C
13 177.119 1 1037 631 R H H 1 8.743 1 1038 631 R N N 15 121.276 1
1039 632 K CB C 13 32.019 1 1040 632 K CO C 13 178.005 1 1041 632 K
H H 1 7.027 1 1042 632 K N N 15 115.472 1 1043 633 R CA C 13 59.11
1 1044 633 R CB C 13 30.377 1 1045 633 R H H 1 8.483 1 1046 633 R N
N 15 116.56 1 1047 634 M CA C 13 57.95 1 1048 634 M CB C 13 31.937
1 1049 634 M H H 1 8.212 1 1050 634 M N N 15 116.933 1 1051 635 V
CA C 13 66.729 1 1052 635 V CB C 13 30.848 1 1053 635 V CG1 C 13
22.182 1 1054 635 V CG2 C 13 24.307 1 1055 635 V CO C 13 176.781 1
1056 635 V H H 1 7.401 1 1057 635 V HG1 H 1 0.511 1 1058 635 V HG2
H 1 1.078 1 1059 635 V N N 15 117.807 1 1060 636 W CA C 13 63.021 1
1061 636 W CB C 13 28.855 1 1062 636 W CO C 13 177.991 1 1063 636 W
H H 1 6.945 1 1064 636 W HE1 H 1 10.208 1 1065 636 W N N 15 117.761
1 1066 636 W NE1 N 15 131.254 1 1067 637 E CA C 13 59.634 1 1068
637 E CB C 13 29.54 1 1069 637 E CO C 13 179.549 1 1070 637 E H H 1
8.942 1 1071 637 E N N 15 118.312 1 1072 638 I CA C 13 65.061 1
1073 638 I CB C 13 36.564 1 1074 638 I CO C 13 178.793 1 1075 638 I
H H 1 8.516 1 1076 638 I N N 15 118.151 1 1077 639 L CA C 13 57.489
1 1078 639 L CB C 13 40.751 1 1079 639 L CD1 C 13 25.065 1 1080 639
L CD2 C 13 22.821 1 1081 639 L H H 1 8.021 1 1082 639 L HD1 H 1
0.577 1 1083 639 L HD2 H 1 0.498 1 1084 639 L N N 15 119.857 1 1085
640 H CA C 13 55.633 1 1086 640 H CB C 13 26.845 1 1087 640 H H H 1
7.758 1 1088 640 H N N 15 112.218 1 1089 641 R CA C 13 56.955 1
1090 641 R CB C 13 25.944 1 1091 641 R CO C 13 174.773 1 1092 641 R
H H 1 7.879 1 1093 641 R N N 15 122.564 1 1094 642 K CA C 13 54.648
1 1095 642 K CB C 13 34.915 1 1096 642 K CO C 13 172.886 1 1097 642
K H H 1 8.22 1 1098 642 K N N 15 121.148 1 1099 643 L CA C 13
53.678 1 1100 643 L CB C 13 41.237 1 1101 643 L CD1 C 13 27.306 1
1102 643 L CD2 C 13 24.45 1 1103 643 L CO C 13 177.594 1 1104 643 L
H H 1 8.136 1 1105 643 L HD1 H 1 0.395 1 1106 643 L HD2 H 1 0.572 1
1107 643 L N N 15 122.205 1 1108 644 L CA C 13 55.222 1 1109 644 L
CB C 13 41.615 1 1110 644 L CO C 13 182.082 1 1111 644 L H H 1
8.863 1 1112 644 L N N 15 130.114 1 *referenced using DSS
(4,4-dimethyl-4-silapentane-1-sulfonic acid) as the H-1 standard
with IUPAC-IUB recommended chemical shift referencing ratios. See,
Wishart, et al., "1H, 13C and 15N Chemical Shift Referencing in
Biomolecular NMR," J. Biomol. NMR 6: 135-140 (1995); and Markley et
al., "Recommendations for the Presentation of NMR Structures of
Proteins and Nucleic Acids,". Pure & Appl. Chem. 70: 117-142
(1998).
TABLE-US-00004 TABLE 4 SENP1 C603S-SUMO.sub.1-92 NMR Chemical Shift
Values. Chemical Shift Ambiguity Index Value Definitions The values
other than 1 are used for those atoms with different chemical
shifts that cannot be assigned to stereospecific atoms or to
specific residues or chains. Index Value Definition 1 Unique
(including isolated methyl protons germinal atoms, and geminal
methyl groups with identical chemical shifts (e.g. ILE HD11, HD12,
HD13 protons) 2 Ambiguity of geminal atoms or geminal methyl proton
groups (e.g. ASP HB2 and HB3 protons, LEU CD1 and CD2 carbons, or
LEU HD11, HD12, HD13 and HD21, HD22, HD23 methyl protons) 3
Aromatic atoms on opposite sides of symmetrical rings (e.g. TYR HE1
and HE2 protons) 4 Intraresidue ambiguities (e.g. LYS HG and HD
protons or TRP HZ2 and HZ3 protons) 5 Interresidue ambiguities (LYS
12 vs. LYS 27) 6 Intermolecular ambiguities (e.g. ASP 31 CA in
monomer 1 and ASP 31 CA in monomer 2 of an asymmetrical homodimer,
duplex DNA assignments, or other assignments that may apply to
atoms in one or more molecule in the molecular assembly) 9
Ambiguous, specific ambiguity not defined Chemical Atom Residue
Amino Atom Atom Iso- shift Unique- number number acid context type
type (ppm)* ness 1 419 E H H 1 7.974 1 2 419 E N N 15 121.157 1 3
420 F H H 1 7.947 1 4 420 F N N 15 119.83 1 5 422 E H H 1 8.562 1 6
422 E N N 15 125.045 1 7 423 I H H 1 8.443 1 8 423 I N N 15 123.042
1 9 424 T H H 1 7.558 1 10 424 T N N 15 122.22 1 11 425 E H H 1
8.835 1 12 425 E N N 15 122.015 1 13 426 E H H 1 8.343 1 14 426 E N
N 15 119.067 1 15 427 M H H 1 7.295 1 16 427 M N N 15 120.177 1 17
428 E H H 1 8.525 1 18 428 E N N 15 119.639 1 19 429 K H H 1 7.793
1 20 429 K N N 15 119.161 1 21 430 E H H 1 7.247 1 22 430 E N N 15
120.017 1 23 432 K H H 1 8.269 1 24 432 K N N 15 117.252 1 25 433 D
H H 1 7.542 1 26 433 D N N 15 116.02 1 27 434 V CG1 C 13 22.744 1
28 434 V H H 1 7.456 1 29 434 V N N 15 115.391 1 32 434 V HG1 H 1
0.768 1 33 435 F H H 1 7.229 1 34 435 F N N 15 118.53 1 35 436 R H
H 1 7.082 1 36 436 R N N 15 119.966 1 37 437 D H H 1 8.266 1 38 437
D N N 15 120.742 1 39 438 G H H 1 7.994 1 40 438 G N N 15 110.561 1
41 439 D H H 1 8.68 1 42 439 D N N 15 121.505 1 43 440 Q H H 1
8.954 1 44 440 Q N N 15 126.9 1 45 441 D H H 1 7.858 1 46 441 D N N
15 115.853 1 47 442 E H H 1 7.08 1 48 442 E N N 15 122.689 1 49 443
V CG1 C 13 21.595 1 50 443 V CG2 C 13 22.122 1 51 443 V H H 1 8.563
1 52 443 V N N 15 128.078 1 55 443 V HG1 H 1 0.731 1 58 443 V HG2 H
1 0.896 1 59 444 L CD1 C 13 27.192 1 60 444 L H H 1 8.928 1 61 444
L N N 15 128.342 1 64 444 L HD1 H 1 0.609 1 65 445 S H H 1 7.327 1
66 445 S N N 15 112.548 1 67 446 E H H 1 7.889 1 68 446 E N N 15
125.959 1 69 447 A H H 1 8.267 1 70 447 A N N 15 125.511 1 71 448 F
H H 1 8.549 1 72 448 F N N 15 117.045 1 73 449 R H H 1 8.43 1 74
449 R N N 15 112.963 1 75 450 L CD1 C 13 26.152 1 76 450 L CD2 C 13
23.171 1 77 450 L H H 1 8.288 1 78 450 L N N 15 121.64 1 81 450 L
HD1 H 1 0.918 1 84 450 L HD2 H 1 1.032 1 85 451 T H H 1 8.297 1 86
451 T N N 15 113.505 1 87 452 I H H 1 8.385 1 88 452 I N N 15
124.62 1 89 453 T H H 1 9.666 1 90 453 T N N 15 120.795 1 91 454 R
H H 1 8.168 1 92 454 R N N 15 123.055 1 93 455 K H H 1 8.41 1 94
455 K N N 15 120.473 1 95 456 D H H 1 7.227 1 96 456 D N N 15
118.114 1 97 457 I H H 1 8.102 1 98 457 I N N 15 122.652 1 99 458 Q
H H 1 7.821 1 100 458 Q N N 15 119.174 1 101 459 T H H 1 7.826 1
102 459 T N N 15 114.259 1 103 460 L CD1 C 13 26.138 1 104 460 L
CD2 C 13 26.003 1 105 460 L H H 1 7.18 1 106 460 L N N 15 116.293 1
109 460 L HD1 H 1 0.845 1 112 460 L HD2 H 1 0.954 1 113 461 D H H 1
7.356 1 114 461 D N N 15 121.211 1 115 462 H H H 1 7.659 1 116 462
H N N 15 120.742 1 117 465 W H H 1 8.258 1 118 465 W N N 15 121.504
1 119 465 W HE1 H 1 9.997 1 120 465 W NE1 H 1 130.62 1 121 466 L
CD1 C 13 25.625 1 122 466 L CD2 C 13 23.625 1 123 466 L H H 1 7.529
1 124 466 L N N 15 126.826 1 127 466 L HD1 H 1 0.684 1 130 466 L
HD2 H 1 0.704 1 131 467 D H H 1 6.937 1 132 467 D N N 15 117.87 1
133 468 D H H 1 8.19 1 134 468 D N N 15 115.279 1 135 470 I H H 1
7.581 1 136 470 I N N 15 118.739 1 137 471 I H H 1 6.823 1 138 471
I N N 15 118.333 1 139 472 D H H 1 8.781 1 140 472 D N N 15 116.126
1 141 473 F H H 1 8.248 1 142 473 F N N 15 124.484 1 143 475 M H H
1 8.642 1 144 475 M N N 15 116.267 1 145 476 D H H 1 7.328 1 146
476 D N N 15 118.167 1 147 477 M H H 1 7.588 1 148 477 M N N 15
122.218 1 149 478 L CD1 C 13 27.574 1 150 478 L CD2 C 13 22.365 1
151 478 L H H 1 7.689 1 152 478 L N N 15 121.041 1 155 478 L HD1 H
1 0.707 1 158 478 L HD2 H 1 0.458 1 159 479 M H H 1 7.581 1 160 479
M N N 15 120.182 1 161 480 E H H 1 8.04 1 162 480 E N N 15 123.808
1 163 481 R H H 1 7.872 1 164 481 R N N 15 121.587 1 165 482 S H H
1 7.133 1 166 482 S N N 15 114.199 1 167 483 K H H 1 6.896 1 168
483 K N N 15 119.755 1 169 484 E H H 1 8.035 1 170 484 E N N 15
122.017 1 171 485 K H H 1 8.183 1 172 485 K N N 15 122.621 1 173
486 G H H 1 8.674 1 174 486 G N N 15 112.424 1 175 487 L CD1 C 13
25.976 1 176 487 L CD2 C 13 23.426 1 177 487 L H H 1 7.277 1 178
487 L N N 15 122.648 1 181 487 L HD1 H 1 0.802 1 184 487 L HD2 H 1
0.853 1 185 489 S H H 1 9.049 1 186 489 S N N 15 119.012 1 187 490
V CG1 C 13 21.236 1 188 490 V CG2 C 13 23.244 1 189 490 V H H 1
7.325 1 190 490 V N N 15 119.713 1 191 490 V HG1 H 1 0.583 1 196
490 V HG2 H 1 0.912 1 197 491 H H H 1 8.716 1 198 491 H N N 15
125.135 1 199 492 A H H 1 7.396 1 200 492 A N N 15 130.645 1 201
494 D H H 1 8.676 1 202 494 D N N 15 117.371 1 203 495 T H H 1
8.641 1 204 495 T N N 15 112.625 1 205 497 F H H 1 7.868 1 206 497
F N N 15 122.062 1 207 498 F H H 1 9.932 1 208 498 F N N 15 121.373
1 209 499 T H H 1 6.901 1 210 499 T N N 15 113.079 1 211 500 K H H
1 7.689 1 212 500 K N N 15 123.985 1 213 501 L CD1 C 13 21.431 1
214 501 L CD2 C 13 26.226 1 217 501 L HD1 H 1 0.589 1 220 501 L HD2
H 1 0.244 1 221 502 K H H 1 8.034 1 222 502 K N N 15 118.063 1 223
503 T H H 1 7.409 1 224 503 T N N 15 109.905 1 225 504 A H H 1
8.332 1 226 504 A N N 15 125.057 1 227 505 G H H 1 7.268 1 228 505
G N N 15 109.015 1 229 506 Y H H 1 8.399 1 230 506 Y N N 15 118.799
1 231 507 Q H H 1 8.555 1 232 507 Q N N 15 114.738 1 233 508 A H H
1 7.049 1 234 508 A N N 15 118.763 1 235 509 V CG1 C 13 19.211 1
236 509 V CG2 C 13 20.045 1 237 509 V H H 1 6.759 1 238 509 V N N
15 105.237 1 241 509 V HG1 H 1 0.198 1 244 509 V HG2 H 1 0.47 1 245
510 K H H 1 7.119 1 246 510 K N N 15 128.018 1 247 511 R H H 1
8.684 1 248 511 R N N 15 117.231 1 249 512 W H H 1 8.542 1 250 512
W N N 15 120.625 1 251 512 W HE1 H 1 9.942 1 252 512 W NE1 H 1
130.341 1 253 513 T H H 1 7.069 1 254 513 T N N 15 106.003 1 255
514 K H H 1 7.238 1 256 514 K N N 15 121.358 1
257 515 K H H 1 8.487 1 258 515 K N N 15 116.476 1 259 516 V CG1 C
13 22.32 1 260 516 V CG2 C 13 19.555 1 261 516 V H H 1 7.299 1 262
516 V N N 15 119.473 1 265 516 V HG1 H 1 1.018 1 268 516 V HG2 H 1
0.836 1 269 517 D H H 1 8.389 1 270 517 D N N 15 124.991 1 271 518
V CG1 C 13 21.972 1 272 518 V CG2 C 13 17.937 1 273 518 V H H 1
8.864 1 274 518 V N N 15 122.483 1 277 518 V HG1 H 1 0.73 1 280 518
V HG2 H 1 0.268 1 281 519 F H H 1 7.156 1 282 519 F N N 15 111.705
1 283 520 S H H 1 7.418 1 284 520 S N N 15 114.604 1 285 521 V CG1
C 13 21.606 1 286 521 V CG2 C 13 19.292 1 287 521 V H H 1 6.57 1
288 521 V N N 15 114.783 1 291 521 V HG1 H 1 0.698 1 294 521 V HG2
H 1 0.757 1 295 522 D H H 1 8.102 1 296 522 D N N 15 121.096 1 297
523 I H H 1 8.117 1 298 523 I N N 15 118.339 1 299 524 L CD1 C 13
24.558 2 300 524 L CD2 C 13 24.558 2 301 524 L H H 1 9.253 1 302
524 L N N 15 122.892 1 305 524 L HG1 H 1 0.841 2 308 524 L HG2 H 1
0.841 2 309 525 L CD1 C 13 27.708 2 310 525 L CD2 C 13 23.849 1 311
525 L H H 1 8.636 1 312 525 L N N 15 121.574 1 315 525 L HD1 H 1
0.775 1 318 525 L HD2 H 1 0.735 1 319 526 V CG1 C 13 20.514 1 320
526 V CG2 C 13 19.367 1 323 526 V HG1 H 1 -0.557 1 326 526 V HG2 H
1 0.332 1 327 528 I H H 1 8.599 1 328 528 I N N 15 125.838 1 329
529 H H H 1 9.049 1 330 529 H N N 15 130.138 1 331 530 L CD1 C 13
25.956 1 332 530 L CD2 C 13 23.945 1 333 530 L H H 1 8.536 1 334
530 L N N 15 131.927 1 337 530 L HD1 H 1 0.891 1 340 530 L HD2 H 1
0.759 1 341 531 G H H 1 8 1 342 531 G N N 15 116.007 1 343 532 V
CG1 C 13 20.988 1 344 532 V CG2 C 13 17.895 1 347 532 V HG1 H 1
0.507 1 350 532 V HG2 H 1 0.396 1 351 533 H H H 1 7.652 1 352 533 H
N N 15 123.973 1 353 534 W H H 1 7.787 1 354 534 W N N 15 125.281 1
355 534 W HE1 H 1 9.36 1 356 534 W NE1 H 1 128.566 1 357 535 C H H
1 9.374 1 358 535 C N N 15 118.493 1 359 536 L CD1 C 13 22.488 1
360 536 L CD2 C 13 26.383 1 363 536 L HD1 H 1 0.708 1 366 536 L HD2
H 1 0.622 1 367 537 A H H 1 8.735 1 368 537 A N N 15 127.969 1 369
538 V CG1 C 13 21.782 1 370 538 V CG2 C 13 22.071 1 371 538 V H H 1
8.971 1 372 538 V N N 15 120.496 1 375 538 V HG1 H 1 0.885 1 378
538 V HG2 H 1 0.831 1 379 539 V CG1 C 13 20.583 1 380 539 V CG2 C
13 19.613 1 381 539 V H H 1 9.299 1 382 539 V N N 15 131.537 1 385
539 V HG1 H 1 0.452 1 388 539 V HG2 H 1 0.894 1 389 540 D H H 1
8.849 1 390 540 D N N 15 127.571 1 391 541 F H H 1 9.394 1 392 541
F N N 15 125.014 1 393 542 R H H 1 8.636 1 394 542 R N N 15 118.378
1 395 543 K H H 1 6.667 1 396 543 K N N 15 115.058 1 397 544 K H H
1 7.337 1 398 544 K N N 15 116.66 1 399 545 D H H 1 7.143 1 400 545
D N N 15 114.477 1 401 546 I H H 1 8.348 1 402 546 I N N 15 121.213
1 403 547 T H H 1 8.68 1 404 547 T N N 15 122.503 1 405 548 Y H H 1
8.599 1 406 548 Y N N 15 129.81 1 407 549 Y H H 1 8.988 1 408 549 Y
N N 15 126.841 1 409 550 D H H 1 7.986 1 410 550 D N N 15 124.111 1
411 551 S H H 1 9.257 1 412 551 S N N 15 123.506 1 413 552 M H H 1
9.118 1 414 552 M N N 15 122.883 1 415 553 G H H 1 7.765 1 416 553
G N N 15 110.571 1 417 554 G H H 1 7.593 1 418 554 G N N 15 108.922
1 419 555 I H H 1 7.986 1 420 555 I N N 15 119.104 1 421 556 D H H
1 7.676 1 422 556 D N N 15 125.208 1 423 557 D H H 1 8.221 1 424
557 D N N 15 122.087 1 425 558 E H H 1 8.444 1 426 558 E N N 15
121.771 1 427 559 A H H 1 7.431 1 428 559 A N N 15 121.623 1 429
560 C H H 1 6.749 1 430 560 C N N 15 112.908 1 431 561 R H H 1
7.935 1 432 561 R N N 15 121.153 1 433 562 I H H 1 8.494 1 434 562
I N N 15 121.654 1 435 563 L CD1 C 13 26.251 1 436 563 L H H 1 7.51
1 437 563 L N N 15 121.675 1 440 563 L HD1 H 1 0.705 1 441 564 L
CD1 C 13 23.21 1 442 564 L CD2 C 13 25.851 1 443 564 L H H 1 7.927
1 444 564 L N N 15 123.395 1 447 564 L HD1 H 1 0.527 1 450 564 L
HD2 H 1 0.857 1 451 565 Q H H 1 7.814 1 452 565 Q N N 15 117.094 1
453 566 Y H H 1 8.072 1 454 566 Y N N 15 122.424 1 455 567 L CD1 C
13 26.248 1 456 567 L H H 1 7.659 1 457 567 L N N 15 119.901 1 460
567 L HD1 H 1 0.295 1 461 568 K H H 1 7.303 1 462 568 K N N 15
117.193 1 463 569 Q H H 1 7.605 1 464 569 Q N N 15 120.146 1 465
570 E H H 1 8.815 1 466 570 E N N 15 125.031 1 467 571 S H H 1
7.999 1 468 571 S N N 15 113.963 1 469 572 I H H 1 6.963 1 470 572
I N N 15 120.839 1 471 573 D H H 1 8.221 1 472 573 D N N 15 120.607
1 473 574 K H H 1 8.471 1 474 574 K N N 15 110.577 1 475 575 K H H
1 7.695 1 476 575 K N N 15 115.597 1 477 580 D H H 1 7.94 1 478 580
D N N 15 129.176 1 479 581 T H H 1 7.92 1 480 581 T N N 15 115.295
1 481 582 D H H 1 8.477 1 482 582 D N N 15 125.759 1 483 584 W HE1
H 1 10.14 1 484 584 W NE1 H 1 131.131 1 485 585 Q H H 1 8.228 1 486
585 Q N N 15 121.5 1 487 586 L CD1 C 13 24.774 1 488 586 L H H 1
8.179 1 489 586 L N N 15 123.493 1 492 586 L HD1 H 1 0.902 1 493
587 F H H 1 8.927 1 494 587 F N N 15 120.564 1 495 588 S H H 1
8.491 1 496 588 S N N 15 116.853 1 497 589 K H H 1 8.272 1 498 589
K N N 15 125.148 1 499 590 K H H 1 9.035 1 500 590 K N N 15 125.714
1 501 591 S H H 1 8.628 1 502 591 S N N 15 117.949 1 503 592 Q H H
1 7.72 1 504 592 Q N N 15 115.49 1 505 593 E H H 1 8.054 1 506 593
E N N 15 117.488 1 507 594 I H H 1 7.053 1 508 594 I N N 15 112.806
1 509 596 Q H H 1 8.456 1 510 596 Q N N 15 120.73 1 511 597 Q H H 1
8.576 1 512 597 Q N N 15 119.759 1 513 598 M H H 1 9.401 1 514 598
M N N 15 120.151 1 515 599 D H H 1 7.357 1 516 599 D N N 15 116.972
1 517 602 D H H 1 7.383 1 518 602 D N N 15 119.804 1 519 603 S H H
1 8.061 1 520 603 S N N 15 121.228 1 521 604 G H H 1 8.814 1 522
604 G N N 15 109.019 1 523 605 M H H 1 6.87 1 524 605 M N N 15
119.181 1 525 606 F H H 1 8.087 1 526 606 F N N 15 120.065 1 527
607 A H H 1 7.948 1 528 607 A N N 15 119.608 1 529 608 C H H 1
7.095 1 530 608 C N N 15 112.346 1 531 609 K H H 1 7.948 1 532 609
K N N 15 118.448 1 533 610 Y H H 1 9.542 1 534 610 Y N N 15 122.405
1 535 611 A H H 1 7.333 1 536 611 A N N 15 119.249 1 537 612 D H H
1 8.263 1 538 612 D N N 15 120.131 1 539 613 C H H 1 7.203 1 540
613 C N N 15 115.162 1 541 614 I H H 1 8.484 1 542 614 I N N 15
120.137 1 543 615 T H H 1 8.165 1 544 615 T N N 15 109.158 1 545
616 K H H 1 7.126 1 546 616 K N N 15 119.661 1 547 617 D H H 1
7.896 1 548 617 D N N 15 118.551 1 549 618 R H H 1 7.855 1 550 618
R N N 15 117.684 1 551 620 I H H 1 8.365 1 552 620 I N N 15 122.909
1 553 621 D H H 1 7.872 1 554 621 D N N 15 127.17 1 555 622 F H H 1
6.479 1 556 622 F N N 15 114.836 1 557 623 T H H 1 10.893 1 558 623
T N N 15 113.615 1 559 624 Q H H 1 9.761 1 560 624 Q N N 15 119.808
1 561 625 Q H H 1 8.292 1
562 625 Q N N 15 119.007 1 563 626 H H H 1 7.627 1 564 626 H N N 15
117.04 1 565 627 M H H 1 7.554 1 566 627 M N N 15 118.727 1 567 629
Y H H 1 7.389 1 568 629 Y N N 15 120.089 1 569 630 F H H 1 8.629 1
570 630 F N N 15 119.732 1 571 631 R H H 1 8.753 1 572 631 R N N 15
122.645 1 573 632 K H H 1 6.955 1 574 632 K N N 15 116.472 1 575
633 R H H 1 8.412 1 576 633 R N N 15 117.619 1 577 634 M H H 1
8.154 1 578 634 M N N 15 117.932 1 579 635 V CG1 C 13 22.402 1 580
635 V CG2 C 13 24.472 1 581 635 V H H 1 7.322 1 582 635 V N N 15
118.68 1 585 635 V HG1 H 1 0.533 1 588 635 V HG2 H 1 1.092 1 589
636 W H H 1 6.859 1 590 636 W N N 15 118.812 1 591 636 W HE1 H 1
10.124 1 592 636 W NE1 H 1 132.344 1 593 637 E H H 1 8.839 1 594
637 E N N 15 119.355 1 595 638 I H H 1 8.41 1 596 638 I N N 15
119.139 1 597 639 L CD1 C 13 25.313 1 598 639 L CD2 C 13 22.916 1
599 639 L H H 1 7.977 1 600 639 L N N 15 120.642 1 603 639 L HD1 H
1 0.596 1 606 639 L HD2 H 1 0.514 1 607 640 H H H 1 7.669 1 608 640
H N N 15 113.238 1 609 641 R H H 1 7.794 1 610 641 R N N 15 123.547
1 611 642 K H H 1 8.152 1 612 642 K N N 15 122.071 1 613 643 L CD1
C 13 27.478 1 614 643 L CD2 C 13 24.632 1 615 643 L H H 1 8.058 1
616 643 L N N 15 123.331 1 619 643 L HD1 H 1 0.412 1 622 643 L HD2
H 1 0.573 1 623 644 L H H 1 8.748 1 624 644 L N N 15 131.146 1
*referenced using DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid)
as the H-1 standard with IUPAC-IUB recommended chemical shift
referencing ratios. See, Wishart, et al., "1H, 13C and 15N Chemical
Shift Referencing in Biomolecular NMR," J. Biomol. NMR 6: 135-140
(1995); and Markley et al., "Recommendations for the Presentation
of NMR Structures of Proteins and Nucleic Acids,". Pure & Appl.
Chem. 70: 117-142 (1998).
TABLE-US-00005 Sequence Listing Isoform 1 SENP1 SEQ ID NO: 1
MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR QGHLDRSFTC
STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ KSRNSRSLYL
ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS PKKTQRRHVS
TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL KDSLFKNGNS
CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH HPHHHHSVPH
QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD SRARERLRQI
EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG HKLTDSEDEF
PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI INFYMNMLME
RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL GVHWCLAVVD
FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK SQEIPQQMNG
SDCGMFACKY ADCITKDRPI NFTQQHMPYF RKRMVWEILH RKLL Isoform 2 SENP1
SEQ ID NO: 2 MDDIADRMRM DAGEVTLVNH NSVFKTHLLP QTGFPEDQLS LSDQQILSSR
QGHLDRSFTC STRSAAYNPS YYSDNPSSDS FLGSGDLRTF GQSANGQWRN STPSSSSSLQ
KSRNSRSLYL ETRKTSSGLS NSFAGKSNHH CHVSAYEKSF PIKPVPSPSW SGSCRRSLLS
PKKTQRRHVS TAEETVQEEE REIYRQLLQM VTGKQFTIAK PTTHFPLHLS RCLSSSKNTL
KDSLFKNGNS CASQIIGSDT SSSGSASILT NQEQLSHSVY SLSSYTPDVA FGSKDSGTLH
HPHHHHSVPH QPDNLAASNT QSEGSDSVIL LKVKDSQTPT PSSTFFQAEL WIKELTSVYD
SRARERLRQI EEQKALALQL QNQRLQEREH SVHDSVELHL RVPLEKEIPV TVVQETQKKG
HKLTDSEDEF PEITEEMEKE IKNVFRNGNQ DEVLSEAFRL TITRKDIQTL NHLNWLNDEI
INFYMNMLME RSKEKGLPSV HAFNTFFFTK LKTAGYQAVK RWTKKVDVFS VDILLVPIHL
GVHWCLAVVD FRKKNITYYD SMGGINNEAC RILLQYLKQE SIDKKRKEFD TNGWQLFSKK
SQIPQQMNGS DCGMFACKYA DCITKDRPIN FTQQHMPYFR KRMVWEILHR KLL (Isoform
1) C-Terminal Region SENP1 SEQ ID NO: 3
EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND
EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV
FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK
QESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDCGMFACKYADCITKDR
PINFTQQHMPYFRKRMVWEILHRKLL (Isoform 1) C-Terminal Region SENP1
C6035 SEQ ID NO: 4
EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND
EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV
FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK
QESIDKKRKEFDTNGWQLFSKKSQEIPQQMNGSDSGMFACKYADCITKDR
PINFTQQHMPYFRKRMVWEILHRKLL (Isoform 2) C-Terminal Region SENP1 SEQ
ID NO: 5 EFPEITEEMEKEIKNVFRNGNQDEVLSEAFRLTITRKDIQTLNHLNWLND
EIINFYMNMLMERSKEKGLPSVHAFNTFFFTKLKTAGYQAVKRWTKKVDV
FSVDILLVPIHLGVHWCLAVVDFRKKNITYYDSMGGINNEACRILLQYLK
QESIDKKRKEFDTNGWQLFSKKSQIPQQMNGSDCGMFACKYADCITKDRP
INFTQQHMPYFRKRMVWEILHRKLL (Isoform 1) Protease Region 450-613 SENP1
SEQ ID NO: 6 LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFT
KLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYY
DSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMN GSDCGMFACKYADC
(Isoform 1) Protease Region 450-613 SENP1 C6035 SEQ ID NO: 7
LTITRKDIQTLNHLNWLNDEIINFYMNMLMERSKEKGLPSVHAFNTFFFT
KLKTAGYQAVKRWTKKVDVFSVDILLVPIHLGVHWCLAVVDFRKKNITYY
DSMGGINNEACRILLQYLKQESIDKKRKEFDTNGWQLFSKKSQEIPQQMN GSDSGMFACKYADC
SUMO1 SEQ ID NO: 8
MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKES
YCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQEQTGGHST V SUMO1 (1-92)
SEQ ID NO: 9 MSDQEAKPSTEDLGDKKEGEYIKLKVIGQDSSEIHFKVKMTTHLKKLKES
YCQRQGVPMNSLRFLFEGQRIADNHTPKELGMEEEDVIEVYQ
EMBODIMENTS
Embodiment 1
A method of detecting binding of an SENP1 polypeptide to a
compound, the method comprising:
(i) contacting an SENP1 polypeptide with a compound;
(ii) allowing the compound to bind to the SENP1 polypeptide,
thereby forming a SENP1-compound complex;
(iii) detecting the SENP1-compound complex using nuclear magnetic
resonance, thereby detecting binding of the SENP1 polypeptide to
the compound.
Embodiment 2
The method of embodiment 1, wherein the detecting comprises
determining a chemical shift for an amino acid in an active site of
the SENP1 polypeptide.
Embodiment 3
The method of embodiment 2, wherein the chemical shift in the
presence of the compound is changed relative to the corresponding
chemical shift in the absence of the compound.
Embodiment 4
The method of embodiment 2 or 3, wherein the amino acid is an amino
acid of SEQ ID NOs: 3, 4, 5, 6 or 7.
Embodiment 5
The method of embodiment 2 or 3, wherein the amino acid is selected
from the group consisting of D550, H533, C603, W465, W534, L466,
G531, C535, M552, G554, E469 and Q596.
Embodiment 6
The method of embodiment 2 or 3, wherein the amino acid is
5603.
Embodiment 7
The method of embodiment 2 or 3, wherein the amino acid is amino
acid residue 440-455, 463-473, 493-515, 529-535, 550-554, or
596-603 of SEQ ID NO:1.
Embodiment 8
The method of embodiment 1, wherein the SENP1 polypeptide comprises
SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
Embodiment 9
The method of embodiment 1, wherein the SENP1 polypeptide comprises
amino acid residue 603 of SEQ ID NO:1.
Embodiment 10
The method of embodiment 9, wherein the SENP1 polypeptide comprises
a mutation at amino acid residue 603 of SEQ ID NO:1.
Embodiment 11
The method of embodiment 10, wherein the mutation is C603S.
Embodiment 12
The method of embodiment 1, wherein the SENP1 polypeptide comprises
amino acid residues 440-455, 463-473, 493-515, 529-535, 550-554, or
596-603 of SEQ ID NO:1.
Embodiment 13
The method of any one of embodiments 1-12, wherein the SENP1 or
SENP1-compound complex is bound to a SUMO protein thereby forming a
SENP1-SUMO complex or SENP1-SUMO-compound complex.
Embodiment 14
The method of embodiment 13, wherein the SUMO protein is a
truncated SUMO protein.
Embodiment 15
The method of embodiment 2, wherein the active site is a
catalytically active site.
Embodiment 16
The method of embodiment 2, wherein the active site is a site that
binds to the SUMO protein.
Embodiment 17
The method of any one of embodiments 1-16, wherein the compound is
a small molecule.
Embodiment 18
The method of any one of embodiments 1 or 8-17, wherein the
detecting comprises producing an NMR spectra of the SENP1-compound
complex and identifying a change in the NMR spectra relative to the
absence of the compound.
Embodiment 19
The method of embodiment 18, wherein the change is a change in the
chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.
Embodiment 20
The method of embodiment 18, wherein the change is a change in the
chemical shift of an amino acid selected from the group consisting
of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469
and Q596.
Embodiment 21
The method of embodiment 18, wherein the change is a change in the
chemical shift of the amino acid 5603.
Embodiment 22
The method of embodiment 18, wherein the change is a change in the
chemical shift of an amino acid residue 440-455, 463-473, 493-515,
529-535, 550-554, or 596-603 of SEQ ID NO:1.
Embodiment 23
An aqueous composition comprising an SENP1 polypeptide at a pH from
about 6.0 to about 7.5.
Embodiment 24
The aqueous composition of embodiment 23, wherein the pH is about
6.8.
Embodiment 25
The aqueous composition of embodiment 23 or 24, further comprising
a buffering agent, reducing agent, solvent, a base, or combinations
thereof.
Embodiment 26
The aqueous composition of any one of embodiments 23-25, further
comprising sodium phosphate, dimethyl sulfoxide, D2O, sodium azide,
dithiothreitol or combinations thereof.
Embodiment 27
The aqueous composition of embodiment 26, wherein the sodium
phosphate is present at about 20 mM.
Embodiment 28
The aqueous composition of any one of embodiments 23-27, wherein
the SENP1 polypeptide comprises SEQ ID NO:1, 2, 3, 4, 5, 6, or
7.
Embodiment 29
The aqueous composition of any one of embodiments 23-27, wherein
the SENP1 polypeptide comprises amino acid residues 440-455,
463-473, 493-515, 529-535, 550-554, or 596-603 numbered relative to
SEQ ID NO:1.
Embodiment 30
The aqueous composition of any one of embodiments 23-29, wherein
the SENP1 polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO complex.
Embodiment 31
The aqueous composition of any one of embodiments 23-29, wherein
the SENP1 polypeptide is bound to a compound thereby forming a
SENP1-compound complex.
Embodiment 32
The aqueous composition of embodiment 31, wherein the SENP1
polypeptide is bound to a SUMO protein thereby forming a
SENP1-SUMO-compound complex.
Embodiment 33
The aqueous composition of embodiment 30 or 32, wherein the SUMO
protein is a truncated SUMO protein.
Embodiment 34
An NMR apparatus comprising an NMR sample container for NMR
analysis, the NMR sample container comprising the aqueous
composition of any one of embodiments 23-33.
Embodiment 35
A method of screening for an inhibitor of SENP1 comprising
contacting a composition comprising an SENP1 polypeptide with a
test compound and detecting whether the test compound binds the
SENP1 polypeptide or fragment thereof by nuclear magnetic
resonance.
Embodiment 36
The method of embodiment 35, wherein the detecting comprises
determining a chemical shift for an amino acid in an active site of
the SENP1 polypeptide.
Embodiment 37
The method of embodiment 36, wherein the amino acid is an amino
acid of SEQ ID NOs: 3, 4, 5, 6 OR 7.
Embodiment 38
The method of embodiment 36, wherein the amino acid is selected
from the group consisting of D550, H533, C603, W465, W534, L466,
G531, C535, M552, G554, E469 and Q596.
Embodiment 39
The method of embodiment 36, wherein the amino acid is S603.
Embodiment 40
The method of embodiment 36, wherein the amino acid is amino acid
residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of
SEQ ID NO:1.
Embodiment 41
The method of embodiment 35, wherein the SENP1 polypeptide
comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
Embodiment 42
The method of embodiment 35, wherein the SENP1 polypeptide
comprises amino acid residue 603 of SEQ ID NO:1.
Embodiment 43
The method of embodiment 42, wherein the SENP1 polypeptide
comprises a mutation at amino acid residue 603 of SEQ ID NO:1.
Embodiment 44
The method of embodiment 43, wherein the mutation is C603S.
Embodiment 45
The method of embodiment 35, wherein the SENP1 polypeptide
comprises amino acid residues 440-455, 463-473, 493-515, 529-535,
550-554, or 596-603 of SEQ ID NO:1.
Embodiment 46
The method of any one of embodiments 35-45, wherein the SENP1
polypeptide is bound to a SUMO protein thereby forming a SENP1-SUMO
complex.
Embodiment 47
The method of embodiment 46, wherein the SUMO protein is a
truncated SUMO protein.
Embodiment 48
The method of any one of embodiments 35-47, wherein the chemical
shift in the presence of the test compound is changed relative to
the corresponding chemical shift in the absence of the test
compound.
Embodiment 49
The method of any one of embodiments 35-47, wherein the SENP1 binds
the compound forming an SENP1-compound complex and the detecting
comprises producing an NMR spectra of the SENP1-compound complex
and identifying a change in the NMR spectra relative to the absence
of the compound.
Embodiment 50
The method of embodiment 49, wherein the change is a change in the
chemical shift of an amino acid of SEQ ID NOs: 3, 4, 5, 6 or 7.
Embodiment 51
The method of embodiment 49, wherein the change is a change in the
chemical shift of an amino acid selected from the group consisting
of D550, H533, C603, W465, W534, L466, G531, C535, M552, G554, E469
and Q596.
Embodiment 52
The method of embodiment 49, wherein the change is a change in the
chemical shift of the amino acid 5603.
Embodiment 53
The method of embodiment 49, wherein the change is a change in the
chemical shift of an amino acid residue 440-455, 463-473, 493-515,
529-535, 550-554, or 596-603 of SEQ ID NO:1.
Embodiment 54
The method of embodiment 49, wherein the change is a change in the
chemical shift of an amino acid in the active site of SENP1.
Embodiment 55
The method of embodiment 54, wherein the active site is a
catalytically active site.
Embodiment 56
The method of embodiment 54, wherein the active site is a site that
binds to the SUMO protein.
Embodiment 57
The method of any one of embodiments 35-56, wherein the test
compound is a small molecule.
Embodiment 58
The method of any one of embodiments 35-57, wherein the composition
is an aqueous solution.
Embodiment 59
The method of any one of embodiments 35-58, wherein the composition
is at a pH from about 6.0 to about 7.5.
Embodiment 60
The method of embodiment 59, wherein the pH is about 6.8.
Embodiment 61
The method of any one of embodiments 35-60, wherein the composition
further comprises a buffering agent, solvent, reducing agent, a
base, or combinations thereof.
Embodiment 62
The method of any one of embodiments 35-60, further comprising
sodium phosphate, D2O, sodium azide, dimethyl sulfoxide,
dithiothreitol or combinations thereof.
Embodiment 63
The method of embodiment 62, wherein the sodium phosphate is
present at about 20 mM.
Embodiment 64
A method of identifying an SENP1 inhibitor, the method
comprising:
combining an SENP1 polypeptide, a SUMO protein, and a test compound
in a reaction vessel;
allowing the SENP1 polypeptide, SUMO protein and test compound to
form a SENP1-SUMO-compound complex; and detecting the
SENP1-SUMO-compound complex thereby identifying the compound as a
SENP1 inhibitor.
Embodiment 65
The method of embodiment 64, wherein one or more of the SENP1
polypeptide, SUMO protein or test compound is labeled.
Embodiment 66
The method of embodiment 65, wherein the label is a fluorescent
label.
Embodiment 67
The method of any one of embodiments 64-66, wherein the test
compound comprises a fluorescent label.
Embodiment 68
The method of any one of embodiments 64-67, wherein binding is
detected by fluorescent polarization.
Embodiment 69
The method of embodiment 64, wherein binding is detected by
detecting a change in the thermal properties of SENP1.
Embodiment 70
The method of embodiment 69, wherein the thermal property is the
melting temperature of SENP1.
Embodiment 71
The method of any one of embodiments 64-70, wherein the SUMO is a
truncated SUMO protein.
Embodiment 72
The method of any one of embodiments 64-70, wherein the SUMO
comprises amino acid residues 1-92 of the SUMO protein.
Embodiment 73
The method of any one of embodiments 64-70, wherein the SUMO
protein comprises SEQ ID NO:8.
Embodiment 74
The method of any one of embodiments 64-70, wherein the SUMO
protein comprises SEQ ID NO:9.
Embodiment 75
The method of any one of embodiments 64-74, wherein the SENP1
polypeptide comprises SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.
Embodiment 76
The method of any one of embodiments 64-74, wherein the SENP1
polypeptide comprises amino acid residue 603 of SEQ ID NO:1.
Embodiment 77
The method of any one of embodiments 64-74, wherein the SENP1
polypeptide comprises a mutation at amino acid residue 603 of SEQ
ID NO:1.
Embodiment 78
The method of embodiment 77, wherein the mutation is C603S.
Embodiment 79
The method of any one of embodiments 64-74, wherein the SENP1
polypeptide comprises amino acid residues 440-455, 463-473,
493-515, 529-535, 550-554, or 596-603 of SEQ ID NO:1.
Embodiment 80
The method of any one of embodiments 64 or 71-79, wherein the
detecting is performed using nuclear magnetic resonance.
Embodiment 81
The method of embodiment 80, wherein the detecting comprises
producing an NMR spectra of the SENP1-SUMO-compound complex and
identifying a change in the NMR spectra relative to the absence of
the test compound.
Embodiment 82
The method of embodiment 81, wherein the change is a change in the
chemical shift of an amino acid in an active site of the SENP1
polypeptide.
Embodiment 83
The method of embodiment 82, wherein the active site is a
catalytically active site.
Embodiment 84
The method of embodiment 82, wherein the active site is a site that
binds to the SUMO protein.
Embodiment 85
The method of embodiment 82, wherein the amino acid is an amino
acid of SEQ ID NOs: 3, 4, 5, 6 OR 7.
Embodiment 86
The method of embodiment 82, wherein the amino acid is selected
from the group consisting of D550, H533, C603, W465, W534, L466,
G531, C535, M552, G554, E469 and Q596.
Embodiment 87
The method of embodiment 82, wherein the amino acid is S603.
Embodiment 88
The method of embodiment 82, wherein the amino acid is amino acid
residue 440-455, 463-473, 493-515, 529-535, 550-554, or 596-603 of
SEQ ID NO:1.
Embodiment 89
The method of any one of embodiments 64-88, wherein the test
compound is a small molecule.
SEQUENCE LISTINGS
1
91644PRTHomo sapiens 1Met Asp Asp Ile Ala Asp Arg Met Arg Met Asp
Ala Gly Glu Val Thr 1 5 10 15 Leu Val Asn His Asn Ser Val Phe Lys
Thr His Leu Leu Pro Gln Thr 20 25 30 Gly Phe Pro Glu Asp Gln Leu
Ser Leu Ser Asp Gln Gln Ile Leu Ser 35 40 45 Ser Arg Gln Gly His
Leu Asp Arg Ser Phe Thr Cys Ser Thr Arg Ser 50 55 60 Ala Ala Tyr
Asn Pro Ser Tyr Tyr Ser Asp Asn Pro Ser Ser Asp Ser 65 70 75 80 Phe
Leu Gly Ser Gly Asp Leu Arg Thr Phe Gly Gln Ser Ala Asn Gly 85 90
95 Gln Trp Arg Asn Ser Thr Pro Ser Ser Ser Ser Ser Leu Gln Lys Ser
100 105 110 Arg Asn Ser Arg Ser Leu Tyr Leu Glu Thr Arg Lys Thr Ser
Ser Gly 115 120 125 Leu Ser Asn Ser Phe Ala Gly Lys Ser Asn His His
Cys His Val Ser 130 135 140 Ala Tyr Glu Lys Ser Phe Pro Ile Lys Pro
Val Pro Ser Pro Ser Trp 145 150 155 160 Ser Gly Ser Cys Arg Arg Ser
Leu Leu Ser Pro Lys Lys Thr Gln Arg 165 170 175 Arg His Val Ser Thr
Ala Glu Glu Thr Val Gln Glu Glu Glu Arg Glu 180 185 190 Ile Tyr Arg
Gln Leu Leu Gln Met Val Thr Gly Lys Gln Phe Thr Ile 195 200 205 Ala
Lys Pro Thr Thr His Phe Pro Leu His Leu Ser Arg Cys Leu Ser 210 215
220 Ser Ser Lys Asn Thr Leu Lys Asp Ser Leu Phe Lys Asn Gly Asn Ser
225 230 235 240 Cys Ala Ser Gln Ile Ile Gly Ser Asp Thr Ser Ser Ser
Gly Ser Ala 245 250 255 Ser Ile Leu Thr Asn Gln Glu Gln Leu Ser His
Ser Val Tyr Ser Leu 260 265 270 Ser Ser Tyr Thr Pro Asp Val Ala Phe
Gly Ser Lys Asp Ser Gly Thr 275 280 285 Leu His His Pro His His His
His Ser Val Pro His Gln Pro Asp Asn 290 295 300 Leu Ala Ala Ser Asn
Thr Gln Ser Glu Gly Ser Asp Ser Val Ile Leu 305 310 315 320 Leu Lys
Val Lys Asp Ser Gln Thr Pro Thr Pro Ser Ser Thr Phe Phe 325 330 335
Gln Ala Glu Leu Trp Ile Lys Glu Leu Thr Ser Val Tyr Asp Ser Arg 340
345 350 Ala Arg Glu Arg Leu Arg Gln Ile Glu Glu Gln Lys Ala Leu Ala
Leu 355 360 365 Gln Leu Gln Asn Gln Arg Leu Gln Glu Arg Glu His Ser
Val His Asp 370 375 380 Ser Val Glu Leu His Leu Arg Val Pro Leu Glu
Lys Glu Ile Pro Val 385 390 395 400 Thr Val Val Gln Glu Thr Gln Lys
Lys Gly His Lys Leu Thr Asp Ser 405 410 415 Glu Asp Glu Phe Pro Glu
Ile Thr Glu Glu Met Glu Lys Glu Ile Lys 420 425 430 Asn Val Phe Arg
Asn Gly Asn Gln Asp Glu Val Leu Ser Glu Ala Phe 435 440 445 Arg Leu
Thr Ile Thr Arg Lys Asp Ile Gln Thr Leu Asn His Leu Asn 450 455 460
Trp Leu Asn Asp Glu Ile Ile Asn Phe Tyr Met Asn Met Leu Met Glu 465
470 475 480 Arg Ser Lys Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn
Thr Phe 485 490 495 Phe Phe Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala
Val Lys Arg Trp 500 505 510 Thr Lys Lys Val Asp Val Phe Ser Val Asp
Ile Leu Leu Val Pro Ile 515 520 525 His Leu Gly Val His Trp Cys Leu
Ala Val Val Asp Phe Arg Lys Lys 530 535 540 Asn Ile Thr Tyr Tyr Asp
Ser Met Gly Gly Ile Asn Asn Glu Ala Cys 545 550 555 560 Arg Ile Leu
Leu Gln Tyr Leu Lys Gln Glu Ser Ile Asp Lys Lys Arg 565 570 575 Lys
Glu Phe Asp Thr Asn Gly Trp Gln Leu Phe Ser Lys Lys Ser Gln 580 585
590 Glu Ile Pro Gln Gln Met Asn Gly Ser Asp Cys Gly Met Phe Ala Cys
595 600 605 Lys Tyr Ala Asp Cys Ile Thr Lys Asp Arg Pro Ile Asn Phe
Thr Gln 610 615 620 Gln His Met Pro Tyr Phe Arg Lys Arg Met Val Trp
Glu Ile Leu His 625 630 635 640 Arg Lys Leu Leu 2643PRTHomo sapiens
2Met Asp Asp Ile Ala Asp Arg Met Arg Met Asp Ala Gly Glu Val Thr 1
5 10 15 Leu Val Asn His Asn Ser Val Phe Lys Thr His Leu Leu Pro Gln
Thr 20 25 30 Gly Phe Pro Glu Asp Gln Leu Ser Leu Ser Asp Gln Gln
Ile Leu Ser 35 40 45 Ser Arg Gln Gly His Leu Asp Arg Ser Phe Thr
Cys Ser Thr Arg Ser 50 55 60 Ala Ala Tyr Asn Pro Ser Tyr Tyr Ser
Asp Asn Pro Ser Ser Asp Ser 65 70 75 80 Phe Leu Gly Ser Gly Asp Leu
Arg Thr Phe Gly Gln Ser Ala Asn Gly 85 90 95 Gln Trp Arg Asn Ser
Thr Pro Ser Ser Ser Ser Ser Leu Gln Lys Ser 100 105 110 Arg Asn Ser
Arg Ser Leu Tyr Leu Glu Thr Arg Lys Thr Ser Ser Gly 115 120 125 Leu
Ser Asn Ser Phe Ala Gly Lys Ser Asn His His Cys His Val Ser 130 135
140 Ala Tyr Glu Lys Ser Phe Pro Ile Lys Pro Val Pro Ser Pro Ser Trp
145 150 155 160 Ser Gly Ser Cys Arg Arg Ser Leu Leu Ser Pro Lys Lys
Thr Gln Arg 165 170 175 Arg His Val Ser Thr Ala Glu Glu Thr Val Gln
Glu Glu Glu Arg Glu 180 185 190 Ile Tyr Arg Gln Leu Leu Gln Met Val
Thr Gly Lys Gln Phe Thr Ile 195 200 205 Ala Lys Pro Thr Thr His Phe
Pro Leu His Leu Ser Arg Cys Leu Ser 210 215 220 Ser Ser Lys Asn Thr
Leu Lys Asp Ser Leu Phe Lys Asn Gly Asn Ser 225 230 235 240 Cys Ala
Ser Gln Ile Ile Gly Ser Asp Thr Ser Ser Ser Gly Ser Ala 245 250 255
Ser Ile Leu Thr Asn Gln Glu Gln Leu Ser His Ser Val Tyr Ser Leu 260
265 270 Ser Ser Tyr Thr Pro Asp Val Ala Phe Gly Ser Lys Asp Ser Gly
Thr 275 280 285 Leu His His Pro His His His His Ser Val Pro His Gln
Pro Asp Asn 290 295 300 Leu Ala Ala Ser Asn Thr Gln Ser Glu Gly Ser
Asp Ser Val Ile Leu 305 310 315 320 Leu Lys Val Lys Asp Ser Gln Thr
Pro Thr Pro Ser Ser Thr Phe Phe 325 330 335 Gln Ala Glu Leu Trp Ile
Lys Glu Leu Thr Ser Val Tyr Asp Ser Arg 340 345 350 Ala Arg Glu Arg
Leu Arg Gln Ile Glu Glu Gln Lys Ala Leu Ala Leu 355 360 365 Gln Leu
Gln Asn Gln Arg Leu Gln Glu Arg Glu His Ser Val His Asp 370 375 380
Ser Val Glu Leu His Leu Arg Val Pro Leu Glu Lys Glu Ile Pro Val 385
390 395 400 Thr Val Val Gln Glu Thr Gln Lys Lys Gly His Lys Leu Thr
Asp Ser 405 410 415 Glu Asp Glu Phe Pro Glu Ile Thr Glu Glu Met Glu
Lys Glu Ile Lys 420 425 430 Asn Val Phe Arg Asn Gly Asn Gln Asp Glu
Val Leu Ser Glu Ala Phe 435 440 445 Arg Leu Thr Ile Thr Arg Lys Asp
Ile Gln Thr Leu Asn His Leu Asn 450 455 460 Trp Leu Asn Asp Glu Ile
Ile Asn Phe Tyr Met Asn Met Leu Met Glu 465 470 475 480 Arg Ser Lys
Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn Thr Phe 485 490 495 Phe
Phe Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala Val Lys Arg Trp 500 505
510 Thr Lys Lys Val Asp Val Phe Ser Val Asp Ile Leu Leu Val Pro Ile
515 520 525 His Leu Gly Val His Trp Cys Leu Ala Val Val Asp Phe Arg
Lys Lys 530 535 540 Asn Ile Thr Tyr Tyr Asp Ser Met Gly Gly Ile Asn
Asn Glu Ala Cys 545 550 555 560 Arg Ile Leu Leu Gln Tyr Leu Lys Gln
Glu Ser Ile Asp Lys Lys Arg 565 570 575 Lys Glu Phe Asp Thr Asn Gly
Trp Gln Leu Phe Ser Lys Lys Ser Gln 580 585 590 Ile Pro Gln Gln Met
Asn Gly Ser Asp Cys Gly Met Phe Ala Cys Lys 595 600 605 Tyr Ala Asp
Cys Ile Thr Lys Asp Arg Pro Ile Asn Phe Thr Gln Gln 610 615 620 His
Met Pro Tyr Phe Arg Lys Arg Met Val Trp Glu Ile Leu His Arg 625 630
635 640 Lys Leu Leu 3226PRTHomo sapiens 3Glu Phe Pro Glu Ile Thr
Glu Glu Met Glu Lys Glu Ile Lys Asn Val 1 5 10 15 Phe Arg Asn Gly
Asn Gln Asp Glu Val Leu Ser Glu Ala Phe Arg Leu 20 25 30 Thr Ile
Thr Arg Lys Asp Ile Gln Thr Leu Asn His Leu Asn Trp Leu 35 40 45
Asn Asp Glu Ile Ile Asn Phe Tyr Met Asn Met Leu Met Glu Arg Ser 50
55 60 Lys Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn Thr Phe Phe
Phe 65 70 75 80 Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala Val Lys Arg
Trp Thr Lys 85 90 95 Lys Val Asp Val Phe Ser Val Asp Ile Leu Leu
Val Pro Ile His Leu 100 105 110 Gly Val His Trp Cys Leu Ala Val Val
Asp Phe Arg Lys Lys Asn Ile 115 120 125 Thr Tyr Tyr Asp Ser Met Gly
Gly Ile Asn Asn Glu Ala Cys Arg Ile 130 135 140 Leu Leu Gln Tyr Leu
Lys Gln Glu Ser Ile Asp Lys Lys Arg Lys Glu 145 150 155 160 Phe Asp
Thr Asn Gly Trp Gln Leu Phe Ser Lys Lys Ser Gln Glu Ile 165 170 175
Pro Gln Gln Met Asn Gly Ser Asp Cys Gly Met Phe Ala Cys Lys Tyr 180
185 190 Ala Asp Cys Ile Thr Lys Asp Arg Pro Ile Asn Phe Thr Gln Gln
His 195 200 205 Met Pro Tyr Phe Arg Lys Arg Met Val Trp Glu Ile Leu
His Arg Lys 210 215 220 Leu Leu 225 4226PRTArtificial
SequenceSynthetic polypeptide 4Glu Phe Pro Glu Ile Thr Glu Glu Met
Glu Lys Glu Ile Lys Asn Val 1 5 10 15 Phe Arg Asn Gly Asn Gln Asp
Glu Val Leu Ser Glu Ala Phe Arg Leu 20 25 30 Thr Ile Thr Arg Lys
Asp Ile Gln Thr Leu Asn His Leu Asn Trp Leu 35 40 45 Asn Asp Glu
Ile Ile Asn Phe Tyr Met Asn Met Leu Met Glu Arg Ser 50 55 60 Lys
Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn Thr Phe Phe Phe 65 70
75 80 Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala Val Lys Arg Trp Thr
Lys 85 90 95 Lys Val Asp Val Phe Ser Val Asp Ile Leu Leu Val Pro
Ile His Leu 100 105 110 Gly Val His Trp Cys Leu Ala Val Val Asp Phe
Arg Lys Lys Asn Ile 115 120 125 Thr Tyr Tyr Asp Ser Met Gly Gly Ile
Asn Asn Glu Ala Cys Arg Ile 130 135 140 Leu Leu Gln Tyr Leu Lys Gln
Glu Ser Ile Asp Lys Lys Arg Lys Glu 145 150 155 160 Phe Asp Thr Asn
Gly Trp Gln Leu Phe Ser Lys Lys Ser Gln Glu Ile 165 170 175 Pro Gln
Gln Met Asn Gly Ser Asp Ser Gly Met Phe Ala Cys Lys Tyr 180 185 190
Ala Asp Cys Ile Thr Lys Asp Arg Pro Ile Asn Phe Thr Gln Gln His 195
200 205 Met Pro Tyr Phe Arg Lys Arg Met Val Trp Glu Ile Leu His Arg
Lys 210 215 220 Leu Leu 225 5225PRTHomo sapiens 5Glu Phe Pro Glu
Ile Thr Glu Glu Met Glu Lys Glu Ile Lys Asn Val 1 5 10 15 Phe Arg
Asn Gly Asn Gln Asp Glu Val Leu Ser Glu Ala Phe Arg Leu 20 25 30
Thr Ile Thr Arg Lys Asp Ile Gln Thr Leu Asn His Leu Asn Trp Leu 35
40 45 Asn Asp Glu Ile Ile Asn Phe Tyr Met Asn Met Leu Met Glu Arg
Ser 50 55 60 Lys Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn Thr
Phe Phe Phe 65 70 75 80 Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala Val
Lys Arg Trp Thr Lys 85 90 95 Lys Val Asp Val Phe Ser Val Asp Ile
Leu Leu Val Pro Ile His Leu 100 105 110 Gly Val His Trp Cys Leu Ala
Val Val Asp Phe Arg Lys Lys Asn Ile 115 120 125 Thr Tyr Tyr Asp Ser
Met Gly Gly Ile Asn Asn Glu Ala Cys Arg Ile 130 135 140 Leu Leu Gln
Tyr Leu Lys Gln Glu Ser Ile Asp Lys Lys Arg Lys Glu 145 150 155 160
Phe Asp Thr Asn Gly Trp Gln Leu Phe Ser Lys Lys Ser Gln Ile Pro 165
170 175 Gln Gln Met Asn Gly Ser Asp Cys Gly Met Phe Ala Cys Lys Tyr
Ala 180 185 190 Asp Cys Ile Thr Lys Asp Arg Pro Ile Asn Phe Thr Gln
Gln His Met 195 200 205 Pro Tyr Phe Arg Lys Arg Met Val Trp Glu Ile
Leu His Arg Lys Leu 210 215 220 Leu 225 6164PRTHomo sapiens 6Leu
Thr Ile Thr Arg Lys Asp Ile Gln Thr Leu Asn His Leu Asn Trp 1 5 10
15 Leu Asn Asp Glu Ile Ile Asn Phe Tyr Met Asn Met Leu Met Glu Arg
20 25 30 Ser Lys Glu Lys Gly Leu Pro Ser Val His Ala Phe Asn Thr
Phe Phe 35 40 45 Phe Thr Lys Leu Lys Thr Ala Gly Tyr Gln Ala Val
Lys Arg Trp Thr 50 55 60 Lys Lys Val Asp Val Phe Ser Val Asp Ile
Leu Leu Val Pro Ile His 65 70 75 80 Leu Gly Val His Trp Cys Leu Ala
Val Val Asp Phe Arg Lys Lys Asn 85 90 95 Ile Thr Tyr Tyr Asp Ser
Met Gly Gly Ile Asn Asn Glu Ala Cys Arg 100 105 110 Ile Leu Leu Gln
Tyr Leu Lys Gln Glu Ser Ile Asp Lys Lys Arg Lys 115 120 125 Glu Phe
Asp Thr Asn Gly Trp Gln Leu Phe Ser Lys Lys Ser Gln Glu 130 135 140
Ile Pro Gln Gln Met Asn Gly Ser Asp Cys Gly Met Phe Ala Cys Lys 145
150 155 160 Tyr Ala Asp Cys 7164PRTArtificial SequenceSynthetic
polypeptide 7Leu Thr Ile Thr Arg Lys Asp Ile Gln Thr Leu Asn His
Leu Asn Trp 1 5 10 15 Leu Asn Asp Glu Ile Ile Asn Phe Tyr Met Asn
Met Leu Met Glu Arg 20 25 30 Ser Lys Glu Lys Gly Leu Pro Ser Val
His Ala Phe Asn Thr Phe Phe 35 40 45 Phe Thr Lys Leu Lys Thr Ala
Gly Tyr Gln Ala Val Lys Arg Trp Thr 50 55 60 Lys Lys Val Asp Val
Phe Ser Val Asp Ile Leu Leu Val Pro Ile His 65 70 75 80 Leu Gly Val
His Trp Cys Leu Ala Val Val Asp Phe Arg Lys Lys Asn 85 90 95 Ile
Thr Tyr Tyr Asp Ser Met Gly Gly Ile Asn Asn Glu Ala Cys Arg 100 105
110 Ile Leu Leu Gln Tyr Leu Lys Gln Glu Ser Ile Asp Lys Lys Arg Lys
115 120 125 Glu Phe Asp Thr Asn Gly Trp Gln Leu Phe Ser Lys Lys Ser
Gln Glu 130 135 140 Ile Pro Gln Gln Met Asn Gly Ser Asp Ser Gly Met
Phe Ala Cys Lys 145 150 155
160 Tyr Ala Asp Cys 8101PRTHomo sapiens 8Met Ser Asp Gln Glu Ala
Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys 1 5 10 15 Lys Glu Gly Glu
Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser Ser 20 25 30 Glu Ile
His Phe Lys Val Lys Met Thr Thr His Leu Lys Lys Leu Lys 35 40 45
Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met Asn Ser Leu Arg Phe 50
55 60 Leu Phe Glu Gly Gln Arg Ile Ala Asp Asn His Thr Pro Lys Glu
Leu 65 70 75 80 Gly Met Glu Glu Glu Asp Val Ile Glu Val Tyr Gln Glu
Gln Thr Gly 85 90 95 Gly His Ser Thr Val 100 992PRTHomo sapiens
9Met Ser Asp Gln Glu Ala Lys Pro Ser Thr Glu Asp Leu Gly Asp Lys 1
5 10 15 Lys Glu Gly Glu Tyr Ile Lys Leu Lys Val Ile Gly Gln Asp Ser
Ser 20 25 30 Glu Ile His Phe Lys Val Lys Met Thr Thr His Leu Lys
Lys Leu Lys 35 40 45 Glu Ser Tyr Cys Gln Arg Gln Gly Val Pro Met
Asn Ser Leu Arg Phe 50 55 60 Leu Phe Glu Gly Gln Arg Ile Ala Asp
Asn His Thr Pro Lys Glu Leu 65 70 75 80 Gly Met Glu Glu Glu Asp Val
Ile Glu Val Tyr Gln 85 90
* * * * *